WO2015127365A2

WO2015127365A2 - Calcium-independent sortase a mutants

Info

Publication number: WO2015127365A2
Application number: PCT/US2015/017116
Authority: WO
Inventors: Jessica Ingram; Hidde Ploegh
Original assignee: Whitehead Institute For Biomedical Research
Priority date: 2014-02-21
Filing date: 2015-02-23
Publication date: 2015-08-27
Also published as: WO2015127365A3

Abstract

Disclosed herein are calcium- independent sortase A mutants exhibiting increased catalytic activity compared to wild-type sortase A.

Description

Inventor(s) : Jessica Ingram, Hidde Ploegh Attorney's Docket No.: WIBR- 1 1-WOl

CALCIUM-INDEPENDENT SORTASE A MUTANTS

RELATED APPLICATIONS

This application claims the benefit of U.S.

Application No. 61/943,042, filed on February 21, 2014. The entire teachings of the above application ( s ) are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under R0A1087879 awarded by the National Institutes of Health. The government has certain rights in the invention .

BACKGROUND OF THE INVENTION

Protein engineering is becoming a widely used tool in many areas of protein biochemistry. One engineering method is controlled protein ligation. Native chemical protein ligation relies on efficient preparation of synthetic peptide esters, which can be technically difficult to prepare for many proteins. Recombinant technologies can be used to generate protein-protein fusions, joining the C-terminus of one protein with the N-terrninus of another protein. Intein-based protein ligation systems can also be used to join proteins. A prerequisite for this intein-mediated ligation method is that the target protein is expressed as a correctly folded fusion with the intein, which is often

challenging. The difficulties of conventional native and recombinant ligation technologies significantly limit the application of protein ligation.

The transpeptidation reaction catalyzed by sortases has emerged as a general method for derivatizing proteins with various types of modifications. For conventional sortase modifications, target proteins are engineered to contain a sortase recognition motif (LPXT) near their C- termini . When incubated with synthetic peptides

containing one or more N-terminal glycine residues and a recombinant sortase, these artificial sortase substrates undergo a transacylation reaction resulting in the exchange of residues C-terminal to the threonine residue with the synthetic oligoglycine peptide , resulting in the protein C-terminus being ligated to the N- terminus of the synthetic peptide .

S . aureus sortase A (SaSrtA) is a common sortase utilized for conventional sortase modification of target proteins . However, SaSrtA possesses a Ca²⁺ dependency that can limit the effectiveness of SaSrtA in low Ca²⁺ concentrations , such as the cytoplasm or when Ca²⁺ binding compounds, for example phosphate, carbonate, and

ethylenediaminetetraacetic acid (EDTA) , are present .

SUMMARY OF THE INVENTION

In some aspects, the disclosure provides a sortase A mutant comprising at least three amino acid substitutions relative to a wild- type sortase A, wherein the amino acid substitutions comprise a) a K residue at position 105 ; b) a Q or A residue at position 108 ; and c) at least one amino acid substitution selected from the group

consisting of i) a R residue at position 94; ii) a S residue at position 94; iii) a N residue at position 160; iv) a A residue at position 165; v) a E residue at position 190; and vi) a T residue at position 196.

In some embodiments, the wild-type sortase A comprises a S. aureus sortase A In some embodiments, the wild-type sortase A comprises a protein sequence of SEQ ID NO: 1. In some embodiments, the wild-type sortase A comprises a protein sequence of SEQ ID NO: 3. In some embodiments, the wild- type sortase A comprises a protein of SEQ ID NO: 5.

In some embodiments, the mutant comprises a deletion of amino acids 2-25. In some embodiments, comprising a deletion of amino acids 2-59.

In some embodiments, the mutant comprises at least two amino acid substitutions selected from the group consisting of i) -vi) . In some embodiments, the mutant comprises at least three amino acid substitutions selected from the group consisting of i) -vi) . In some embodiments, the mutant comprises at least four amino acid substitu ions selected from the group consisting of i) -vi) . In some embodiments, the mutant comprises at least five amino acid substitutions selected from the group consisting of i) -vi) .

In some embodiments, the mutant comprises at least 60% sequence identity to amino acid residues 60-206 of the wild-type sortase A. In some embodiments, the mutant comprises at least 80% sequence identity to amino acid residues 60-206 of the wild-type sortase A. In some embodiments, the mutant comprises at least 90% sequence identity to amino acid residues 60-206 of the wild-type sortase A. In some embodiments, the mutant comprises one or more C- terminal or N-terminal tags. In some embodiments, the one or more C-terminal or N-terminal tags comprises a His6 tag.

In some embodiments, the mutant exhibits sortase A catalytic activity in the absence of calcium.

In some embodiments, the mutant exhibits sortase A catalytic activity in the absence of exogenous calcium. In some embodiments, the mutant exhibits sortase A catalytic activity in the presence of calcium-binding proteins. In some embodiments, the mutant exhibits sortase A catalytic activity in the presence of calcium concentrations up to 10 m .

In some aspects, the disclosure provides a

polynucleotide encoding a mutant srtA described herein.

In some aspects, the disclosure provides a polynucleotide encoding a catalytically active variant or fragment of a mutant srtA described herein.

In some aspects , the disclosure provides a nucleic acid construct comprising the polynucleotides described herein .

In some aspects, the disclosure provides a host cell transformed with the nucleic acid constructs described herein .

In some aspects, the disclosure provides a method of preparing a mutant sortase A comprising: (a) culturing the host cell of claim 22 in a suitable culture medium under suitable conditions to produce the mutant sortase A; and optionally (b) purifying the mutant sortase A to provide a mutant sortase A.

In some aspects, the disclosure provides an enzyme composition comprising at least one sortase A mutant described herein. In some aspects, the disclosure provides a method comprising performing a sortase-mediated transpeptidation reaction catalyzed by the enzyme compositions described herein .

In some aspects, the disclosure relates to the use of an enzyme composition described herein for the sortagging of a target protein.

In some aspects, the disclosure provides a method for sortagging a target protein, comprising: (a)

providing a target protein comprising a sortase

recognition motif; (b) providing a moiety conjugated to a terminal oligoglycine sequence or a terminal alkylamine ; and (c) contacting the target protein with the moiety in the presence of the enzyme composition of claim 24 under conditions suitable for the sortase A mutant to

transamidate the target protein and the moiety, thereby sortagging the target protein.

In some embodiments, the terminal oligoglycine sequence comprises 1-10 N-terminal glycine residues.

In some embodiments, the moiety comprises an amino acid, a peptide, a protein, a polynucleotide, a

carbohydrate, a tag, a metal atom, a chelating agent, a contrast agent, a catalyst, a polymer, a recognition element, a small molecule, a lipid, a label, an epitope, a small molecule, a therapeutic agent, a cross- linker, a toxin, a radioisotope, an antigen, or a click chemistry handle .

In some aspects, the disclosure provides a kit for sortagging a target protein comprising the enzyme composition of claim 24.

In some aspects, the disclosure provides a sortase A mutant comprising an amino acid sequence at least 80% identical to SEQ ID NO : 9, and wherein the mutant comprises a) a K residue at position 47 of SEQ ID NO: 9; b) a Q or A residue at position 50 of SEQ ID NO: 9; and c) at least one amino acid residue of SEQ ID NO: 9 selected from the group consisting of i) a R residue at position 36 of SEQ ID NO : 9 ; ii) a N residue at position 102 of SEQ ID NO: 9; iii) a A residue at position 107 of SEQ ID NO: 9; iv) a E residue at position 132 of SEQ ID NO: 9; and v) a T residue at position 138 of SEQ ID NO: 9.

In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 16. In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 17. In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 18. In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 19. In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 20. In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 21.

In some embodiments, the mutant comprises at least two amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) . In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 15.

In some embodiments, the mutant comprises at least three amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) . In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 13. In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 14.

In some embodiments, the mutant comprises at least four amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) . In some embodiments , the mutant comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, the mutant comprises an amino acid sequence at least 90% identical to SEQ ID NO: 9. In some embodiments, the mutant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 9. In some embodiments, the mutant comprises an amino acid sequence at least 96% identical to SEQ ID NO: 9. In some

embodiments, the mutant comprises an amino acid sequence at least 97% identical to SEQ ID NO: 9. In some

embodiments, the mutant comprises an amino acid sequence at least 98% identical to SEQ ID NO: 9. In some

embodiments, the mutant comprises an amino acid sequence at least 99% identical to SEQ ID NO: 9. In some

embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 9.

In some aspects, the disclosure comprises an enzyme composition comprising at least one mutant sortase A described herein. In some embodiments, the at least one mutant sortase A is selected from the group consisting of SEQ ID NOs : 9-21. In some embodiments, the at least one mutant sortase A is selected from the group consisting of catalytically active variants of SEQ ID NOs : 9-21. In some embodiments, the at least one mutant sortase A is selected from the group consisting of catalytically active fragments of SEQ ID NOs : 9-21.

In some aspects, the disclosure provides a method comprising performing a sortase-mediated transpeptidation reaction catalyzed an enzyme composition described herein .

In some aspects, the disclosure relates to a method for sortagging a target protein, comprising: (a) providing a target protein comprising a sortase

recognition motif; (b) providing a moiety conjugated to at least one of an oligoglycine sequence or a terminal alkylamine; and (c) contacting the target protein with the moiety in the presence of an enzyme composition described herein under conditions suitable for the sortase A mutant to transamidate the target protein and the moiety, thereby sortagging the target protein.

In some embodiments, the oligoglycine sequence comprises 1-10 N-terminal glycine residues.

In some aspects, the disclosure provides a kit for sortagging a target protein comprising an enzyme composition described herein.

In some aspects, the disclosure provides a

polynucleotide encoding a sortase A mutant comprising a nucleotide sequence at least 80% identical to SEQ ID NO. 10, wherein the nucleotide sequence encodes a) a K residue at position 47 of SEQ ID NO: 9; b) a Q or A residue at position 50 of SEQ ID NO: 9; and c) at least one amino acid residue of SEQ ID NO: 9 selected from the group consisting of i) a R residue at position 36; ii) a N residue at position 102; iii) a A residue at position 107; iv) a E residue at position 132; and v) a T residue at position 138. In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) . In some embodiments, the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9 selected from the group

consisting of i) -v) . In some embodiments, the

polynucleotide encodes at least four amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) - v) .

In some embodiments, the polynucleotide comprises a nucleotide sequence at least 85% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 96% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 97% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 98% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 99% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence of SEQ ID NO : 10.

In some aspects, the disclosure provides a nucleic acid construct comprising any of the polynucleotides described herein. In some embodiments, the nucleic acid construct comprises a nucleotide sequence that encodes one or more C-terminal or N-terminal tags. In some embodiments , the nucleic acid construct comprises one or more C-terminal or N-terminal tags comprises a His6 tag . In some aspects, the disclosure provides a host cell transformed with a nucleic acid construct described herein .

In some aspects, the disclosure provides a method of preparing a mutant sortase A comprising: (a) culturing the host cell of claim 75 in a suitable culture medium under suitable conditions to produce the mutant sortase A; and optionally (b) purifying the mutant sortase A to provide a mutant sortase A.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 is a schematic representation of a sortase- catalyzed transacylation reaction.

FIG . 2 is a schematic representation of the site- specific C-terminal labeling scheme (left) and N-terminal labeling scheme (right) using sortase A. Such labeling begins with a substrate- recognition step (top) , and then proceeds with generation of a thioacyl intermediate (middle) , followed by ligation of an exogenously

introduced nucleophile to form a new peptide bond

(bottom) (Popp and Ploegh, Angew. Chem. Int. Ed. 2011, 50, 5024-5032) .

FIGS . 3A-3H are schematic representations of various polypeptide conjugations using sortase-mediated ligation .

FIG . 3A is a schematic representation showing the sortagging of a molecular probe carrying an oligoglycine tag to a target pro ein having a C- erminal LPXTG- tag . FIG. 3B is a schematic representation showing sortase-mediated ligation of a polypeptide to a nucleic acid .

FIG. 3C is a schematic representation of a sortase- mediated ligation being used to produce a neoglyconjugate by fusing a polypeptide comprising the LPXTG sortase recognition motif to amino-methylene groups in 6- aminohexoses .

FIG. 3D is a schematic representation of the immobilization of an LPXTG- tagged protein onto an oligoglycine-coated solid surface, for example,

polystyrene beads or a biosensor chip .

FIG. 3E is a schematic representation of a cell surface protein genetically engineered to include an extracellular C-terminal region expressing a C-terminal LPXTG motif, which has been labeled with a triglycine- tagged probe by sortagging.

FIG. 3F is a schematic illustration of the

dimerization/oligomerization of a protein by sortagging of a bifunctional protein possessing an N-terminal oligoglycine tag and a C-terminal LPXTG tag.

FIG . 3G is a schematic representation of the circularization of a bifunctional protein containing an N-terminal oligoglycine tag and a C-terminal LPXTG tag utilizing sortase-mediated ligation.

FIG . 3H is a schematic representation of the site- specific attachment of a lipid utilizing sortase-mediated transpeptidation .

FIGS . 4A-4U show exemplary sequences contemplated by the present disclosure . Mutations relative to wild- type sortase A are shown in red font . Portions of sequences that are absent in an exemplary wild-type S. aureus sortase A sequence and/or contain a 6XHis tag are highlighted in yellow .

FIG. 4A is an exemplary wild type S. aureus Sortase A (SaSrtA) amino acid sequence.

FIG. 4B is an exemplary nucleotide sequence encoding the amino acid sequence shown in FIG. 4A.

FIG. 4C is an exemplary wild type S. aureus Sortase A (SaSrtA) amino acid sequence.

FIG. 4D is an exemplary nucleotide sequence encoding the amino acid sequence shown in FIG. 4C.

FIG. 4E is an exemplary wild type sortase delta 25 amino acid sequence.

FIG. 4F is an exemplary nucleotide sequence encoding the amino acid sequence shown in FIG. 4E.

FIG. 4G is an exemplary mutant srtA

P94R/D160ND165A/K190E/K196T amino acid sequence.

FIG. 4H is an exemplary nucleotide sequence encoding the amino acid sequence shown in FIG. 4G.

FIG. 41 is an exemplary calcium- independent mutant srtA P94R/D160N/D165A/K190E/K196T amino acid sequence.

FIG. 4J is an exemplary nucleotide sequence encoding the amino acid sequence shown in FIG . 41.

FIG . 4K is an exem lary calcium- independent srtA P94S/D160N/D165A/K196T amino acid sequence.

FIG. 4L is an exemplary calcium-independent srtA

D160N/K190E/K196T amino acid sequence.

FIG. 4M is an exemplary calcium- independent srtA P94S/D160N/K196T amino acid sequence.

FIG. 4N is an exemplary calcium- independent srtA P94S/D160N/D165A amino acid sequence.

FIG . 40 is an. exemplary calcium-independent srtA P94S/D165A amino acid sequence . FIG. 4P is an exemplary calcium-independent srtA P94S amino acid sequence.

FIG. 4Q is an exemplary calcium-independent srtA P94R amino acid sequence.

FIG. 4R is an exemplary calcium- independent srtA

D160N amino acid sequence.

FIG. 4S is an exemplary calcium- independent srtA D165A amino acid sequence.

FIG. 4T is an exemplary calcium- independent srtA K190E amino acid sequence.

FIG. 4U is an exemplary calcium- independent srtA K196T amino acid sequence.

FIGS. 5A and 5B show exemplary alignments of various srtA sequences contemplated by the present disclosure.

FIG. 5A shows an alignment of SEQ ID NO: 1 and SEQ

ID NO: 9, indicating that SEQ ID NO: 9 is at least 65% identical to SEQ ID NO : 1.

FIG. 5B shows an alignment of SEQ ID NO: 7 and SEQ ID NO: 9, indicating that SEQ ID NO: 9 is at least 98% identical to SEQ ID NO: 7.

FIG. 6 shows a comparison of activities of various sortases at 0 °C, in the presence and absence of calcium. 30uM of a substrate (VHH84D4) containing an LPETGG- 6xHis C-terminal tag was incubated in 50mM Tris, pH 7.5, 150mM NaCl , 500uM GGG-TAMRA and lOmM CaC12 or lOmM EGTA with either 5uM (1) WT SrtA delta 25, (2) 5uM pentamutant SrtA or (3) heptamutant SrtA for up to 6 hours at 0°C. The reaction was then visualized by running a sample on a 12% Tris-glycine SDS PAGE gel. Incorporation of GGG-TAMRA label at the C-terminus of the substrate was monitored by a shift to a lower molecular weight upon loss of the 6x His tag (Coomassie stained protein gel , upper panel ) and presence of a TAMRA-labeled product (fluorescent scan of protein gel, lower panel) .

FIGS. 7A - 7C are chromatograms demonstrating that the calcium-independent srtA mutant of SEQ ID NO: 9 exists predominantly in monomeric form (FIG. 7A) compared to the calcium-dependent srtA mutant of SEQ ID NO: 7 which exists in both dimeric and monomeric form (FIG. 7B) , and the calcium-dependent wild-type srtA which also exists in both dimeric and monomeric form (FIG. 7C) .

FIG. 8 is a photograph of a gel demonstrating the high purity of monomeric sortase.

FIG. 9 is a chromatogram demonstrating the stability of monomeric sortase. DETAILED DESCRIPTION OF THE INVENTION

Aspects of the disclosure relate to sortase mutants, and in particular to sortase A mutants that exhibit an indifference to calcium. Work described herein

demonstrates that the calcium-independent sortase A mutants described herein surprisingly and unexpectedly exhibit sortase A catalytic activity (e.g., increased catalytic activity compared to wild type sortase A, ) , and do so in a manner that is independent of the presence or concentration of calcium.

Sortases, sortase-mediated transacylation reactions, and their use in transacylation (sometimes also referred to as transpeptidation) for protein engineering are well known to those of skill in the art (see, e.g., Ploegh et al., International Patent Application PCT/US2010/000274 , and Ploegh et al . , International Patent Application

PCT/US2011/033303, the entire contents of each of which are incorporated herein by reference) . FIG. 1 shows an exemplary transpeptidation reaction catalyzed by sortase, which results in the ligation of species containing a sortase recognition motif (e.g., an LPXT motif or an LPXTG motif) with those bearing an N-terminal

oligoglycine moiety. As is shown in FIG. 2, sortases can be used for both C- terminal (left) and N-terminal (right) site-specific labeling.

Those skilled in the art will appreciate that the sortase transacylation reaction allows for the facile installation of all kinds of substituents at the C- terminus or N-terminus of a suitably modified protein. For example, the sortase reaction can be employed for ligating polypeptides to one another, ligating synthetic peptides to recombinant proteins, linking a reporting molecule to a polypeptide, linking a polypeptide to a label or probe (FIG. 3A) , joining a nucleic acid to a polypeptide (FIG. 3B) , ligating a glycan to a polypeptide (FIG. 3C) , conjugating a polypeptide to a solid support or polymer (FIG. 3D) , site- specific modification of the extracellular C-terminal region of cell surface proteins expressed in living cells (FIG. 3E) , site- specific modification of the extracellular N-terminal region of cell surface proteins expressed in living cells ,

dimerization or oligomerization of polypeptides (FIG . 3F) , circularization of polypeptides (FIG . 3G) , site- specific attachment of lipids to polypeptides (FIG. 3H) , and protein purification (FIG . 31) .

As is demonstrated in FIGS . 3A-3I , sortase A derives its ability to site- specifically modify target proteins by recognition of sortase recognition motifs, such as the motif LPXTG. Other suitable sortase recognition motifs are apparent to the skilled artisan. It will be

appreciated that the terms "recognition motif" and

"recognition sequence" , with respect to sequences recognized by sortase, are used interchangeably. In some embodiments, a recognition sequence further comprises one or more additional amino acids, e.g., at the N or C terminus. Such additional amino acids may provide context that improves the recognition of the recognition motif. Those skilled in the art will appreciate that the term "sortase recognition sequence" may refer to a masked or unmasked sortase recognition sequence.

In some embodiments, a calcium-independent mutant sortase has a different substrate specificity with regard to the sortase recognition motif as compared to a calcium-dependent wild type sortase. Sortases with different substrate specificities with regard to the sortase recognition motif recognize different sortase recognition sequences. For example, Sortases 1 and 2 are said to have different substrate specificities if SRM1 and SRM2 are different sortase recognition motifs and Sortase 1 recognizes SRM1 and Sortase 2 is active in recognizing SRM2 but has little or no activity

recognizing SRM1. In some embodiments the substrate specificities overlap in that one of the two sortases recognizes both SRM1 and SR 2 while the other sortase recognizes only one of the SRMs . In some embodiments the substrate specificities do not overlap, e.g., Sortase 1 recognizes SRM1 but does not recognize SRM2 , and Sortase 2 recognizes SRM2 but does not recognize SRM1. In some embodiments, two calcium- independent sortases may alternately or additionally utilize different

nucleophiles . In some embodiments the nucleophile specificity overlaps while in some embodiments the nucleophile specificity does not overlap.

A sortase with an altered substrate specificity with regard to the sortase recognition motif may be generated by engineering one or more mutations in the sortase, e.g., in a region of the protein that is involved in recognition and/or binding of the sortase recognition motif, e.g., the putative substrate recognition loop (e.g., the loop connecting strands β6 and β7 ( β 6/β7 loop) in SrtA (Val¹⁶¹-Asp^17S) . A crystal structure of S. aureus SrtA and a substrate, illustrating the loops, is

described in Zong, Y., et al . , J Biol Chem. 2004 Jul 23,-279(30) :31383-9.) . In some embodiments, a phage- display, yeast display, or other screen of a mutant sortase library randomized in the substrate recognition loop may be performed, and variants with altered

substrate specificity may be identified. A sortase with an altered nucleophile specificity may be generated by engineering one or more mutations in the sortase, e.g., in a region of the protein that is involved in

recognition and/or binding of the nucleophile, e.g., (e.g., the loop connecting strands β7 and β8 in SrtA) . See In some embodiments, a phage-display screen of a mutant sortase library randomized in the substrate recognition loop may be performed, and variants with altered substrate specificity may be identified.

In some embodiments, a calcium- independent sortase described herein is modified to alter its substrate specificity with regard to the sortase recognition motif and/or its nucleophile specificity by introducing one or more mutations into the sortase. In some embodiments, a calcium-dependent sortase is modified to alter its substrate specificity and/or nucleophile specificity and is rendered calcium- independent by introducing the mutations described herein. It will be appreciated that mutations conferring calcium- independence and altered substrate and/or nucleophile specificity may be engineered individually or sequentially in groups of one or more, in any order or combination that results in a desired sequence. In some embodiments two calcium- independent sortases with different substrate

specificities and/or different nucleophile specificities are derived from the same wild type sortase, e.g., S. aureus SrtA. In some embodiments the substrate

specificities do not overlap. Sortases with different substrate specificities may be used, for example, to introduce two different moieties to a target protein.

For example, a target protein may comprise or be modified to comprise first and second SRMs. Two sortases, each of which specifically recognizes only one of the two SRMs, may be used to conjugate two agents to the protein by reaction with the two SRMs, e.g., at the N- and C- termini . The conjugations may take place in a single reaction vessel or may be performed sequentially. The two agents may be any of the agents described herein, and may be the same or different. For example, a first agent may comprise a toxin and a second agent may comprise a detectable label , e.g., for imaging.

In some embodiments , the disclosure provides a kit comprising two calcium- independent sortases with

different substrate specificities with regard to the sortase recognition motif and/or with regard to

nucleophile specificity . In some embodiments two calcium- independent sortases with different substrate specificities and/or different nucleophile specificities are derived from the same wild type sortase , e.g., S . aureus SrtA. In some embodiments the substrate

specificities and/or nucleophile specificities do not overla . Amino acid sequences of sortase A polypeptides and the nucleotide sequences that encode them are known to those of skill in the art and are disclosed in a number of references cited herein, the entire contents of all of which are incorporated herein by reference. A

"polypeptide" , "peptide" or "protein" refers to a molecule comprising at least two covalently attached amino acids. A polypeptide can be made up of naturally occurring amino acids and peptide bonds and/or synthetic peptidomimetic residues and/or bonds. Polypeptides described herein include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial (e.g., E. coli) , yeast, higher plant, insect and mammalian cells. The disclosure also features biologically active fragments of calcium- independent srtA mutants.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline . Amino acid analogs are compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon bound to hydrogen, a carboxyl group, an amino group, and an R group, e.g., norleucine. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid . Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

Sortase A is a polypeptide having a length of 206 amino acids which typically comprises a hydrophobic N- terminal domain (e.g., residues 1 to about 25) which functions as both a signal peptide and a membrane anchoring domain, a central linker domain (e.g., from about residue 26 to about residue 59) , and a C-terminal catalytic domain (e.g., from about residue 60 to about residue 206) .

An exemplary sortase A is the wild-type S . aureus SrtA. An exemplary amino acid sequence of wild-type S . aureus SrtA is shown in FIG . 4A (SEQ ID NO: 1 ; Gene ID : 1125243 ; NCBI RefSeq Acc . No. NP_375640) FIG. 4B shows an exemplary nucleotide sequence (SEQ ID NO: 2; NCBI

Reference Sequence: NC_002745.2) encoding the wild type SaSrtA protein. Another exemplary sequence of wild- type S. aureus SrtA is shown in FIG. 4C (SEQ ID NO: 3; GenBank Accession number AAD48437 ; NCBI Reference Sequence:

NC_002951.2) . It should be appreciated that wild-type S. aureus SrtA sequences, calcium- independent srtA mutants, and catalytically active fragments or variants thereof disclosed herein may comprise either a K or N at position 57. It should be appreciated that wild-type S. aureus SrtA sequences, calcium- independent srtA mutants, and catalytically active fragments or variants thereof disclosed herein may comprise either an E or G at position 167.

Aspects of the present disclosure relate to calcium- independent sortase A mutants. Such mutants may be produced through processes such as directed evolution, site-specific modification, etc . It should be

appreciated that the calcium- independent srtA mutants disclosed herein can be used in any application in which sortagging is desirable, including for example, the sortase -mediated ligation reactions described in FIGS. 3A-3I .

In some embodiments, the calcium- independent srtA mutants comprise at least one amino acid substitution relative to a wild-type sortase A polypeptide, and catalytically active fragments, catalytically active derivatives, or catalytically active variants thereof, and polynucleotides encoding the same.

As used herein "calcium- independent" refers to the ability of a sortase A enzyme to exhibit catalytic activity in a manner that is independent of the presence of calcium, or independent of the amount of calcium present within a concentration range that is not

detrimental to proper functioning of the enzyme. The calcium- independent srtA mutants disclosed herein can be assayed for their ability to exhibit sortase A catalytic activity in a calcium- independent manner by contacting a target protein comprising a C-terminal sortase

recognition motif with a tagged N-terminal oligoglycine derivative in the presence of the calcium- independent srtA mutant, and in the absence of calcium, and

determining whether the target protein is ligated to the tagged N-terminal oligoglycine derivative by the calcium- independent srtA mutant in the absence of calcium. In some embodiments, the calcium- independent srtA mutants can be assayed for their ability to exhibit sortase A catalytic activity in the presence of calcium

concentrations which are lower than calcium

concentrations which are required for calcium-dependent sortase A to exhibit catalytic activity. As used herein, "calcium-dependent" in connection with a sortase means that the catalytic activity of the sortase relies or depends on the presence and concentration of calcium, such that in the absence of calcium or the absence of a sufficient amount of calcium, the calcium-dependent sortase will not exhibit sortase A catalytic activity or has greatly reduced catalytic activity as compared with its activity when calcium is present in sufficient amounts (e.g., 5 mM - 10 mM) .

The present disclosure contemplates any srtA variants which exhibit sortase A catalytic activity comparable to the wild-type srtA enzyme, as long as they possess calcium- independent sortase A catalytic activity. As used herein in connection with "fragments" ,

"derivatives" and "variants", "catalytically active" refers to sortase A catalytic activity. As used herein, "sortase A catalytic activity" refers to the ability of a sortase to catalyze the cleavage of a polypeptide within a sortase A consensus recognition sequence and ligate the free primary amino group (NH₂-CH₂-) of a oligoglycine sequence to the free C-terminal carboxyl group of the cleaved polypeptide. The term "oligoglycine" refers to a (Gly)_{n s}equence, wherein n is between 1 and about 10, or more preferably between 2 and about 5, and even more preferably 2 or 3 , glycine residues. An "N-terminal" oligoglycine sequence is located at the N-terminus of a polypeptide , such that the polypeptide comprises a free primary amino (NH₂„CH₂_) group at its N- erminus . An "N- terminal" oligoglycine sequence can also include an internal oligoglycine sequence that is capable of forming a oligoglycine sequence under applicable conditions , e.g., by cleavage of an N-terminal peptide sequence by an endogenous host cell enzyme, or by specific proteolytic cleavage in vitro . Sortase A catalytic activity of the calcium- independent srtA mutants disclosed herein can be assayed using methods known in the art. The crystal structure of SrtA complexed with a substrate has been determined allowing catalytic active domains of sortase A proteins from various Gram-positive bacterium to be easily discerned by those of skill in the art, see for example, Y. Zong et al . J. Biol Chem. 2004, 279, 31383-31389, which is incorporated herein by reference.

A "variant" of a particular polypeptide or

polynucleotide has one or more alterations (e.g., additions, substitutions, and/or deletions) with respect to a reference polypeptide or polynucleotide, which may be referred to as the "original polypeptide" or "original polynucleotide", respectively. An addition may be an insertion or may be at either terminus. A variant may be shorter or longer than the reference polypeptide or polynucleotide. The term "variant" encompasses

"fragments" . A "fragment" is a continuous portion of a polypeptide or polynucleotide that is shorter than the reference polypeptide or polynucleotide. In some embodiments a variant comprises or consists of a

fragment.. In some embodiments a fragment or variant is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, or more as long as the reference polypeptide or polynucleotide. In some embodiments a fragment may lack an N-terminal and/or C-terminal portion of a reference polypeptide. For example, a fragment may lack up to 5%, 10%, 15%, 20%, or 25% of the length of the polypeptide from either or both ends. A fragment may be an N-terminal, C-terminal, or internal fragment. In some embodiments a variant polypeptide comprises or consists of at least one domain of a reference polypeptide. In some embodiments a variant polynucleotide hybridizes to a reference polynucleotide under art-recognized stringent conditions, e.g., high stringency conditions, for sequences of the length of the reference polypeptide. In some embodiments a variant polypeptide or polynucleotide comprises or consists of a polypeptide or polynucleotide that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical in sequence to the reference polypeptide or polynucleotide over at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the reference polypeptide or polynucleotide. In some embodiments a variant polypeptide comprises or consists of a polypeptide that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical in sequence to the reference polypeptide over at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the reference polypeptide, with the proviso that, for purposes of computing percent identity, a conservative amino acid substitution is considered identical to the amino acid it replaces . In some embodiments a variant polypeptide comprises or consists of a polypeptide that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical to the reference polypeptide over at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the reference polypeptide, with the proviso that any one or more amino acid substitutions (up to the total number of such substitutions) may be restricted to conservative substitutions. In some embodiments a percent identity is measured over at least 100; 200; 300; 400; 500; 600; 700; 800; 900; 1,000; 1,200; 1,500; 2,000; 2,500; 3,000;

3,500; 4,000; 4,500; or 5,000 amino acids. In some embodiments the sequence of a variant polypeptide comprises or consists of a sequence that has N amino acid differences with respect to a reference sequence, wherein N is any integer between 1 and 10 or between 1 and 20 or any integer up to 1%, 2%, 5%, or 10% of the number of amino acids in the reference polypeptide, where an "amino acid difference" refers to a substitution, insertion, or deletion of an amino acid. In some embodiments a difference is a conservative substitution. Conservative substitutions may be made, e.g., on the basis of

similarity in side chain size, polarity, charge,

solubility, hydrophobic!ty, hydrophilicity and/or the amphipathic nature of the residues involved. Suitable conservative substitutions are apparent to the skilled artisa .

In some embodiments a variant is a functional variant, i.e., the variant at least in part retains at least one activity (e.g., calcium- independent sortase A catalytic activity) of the reference polypeptide or polynucleotide. In some embodiments a variant at least in part retains more than one or substantially all known activities of the reference polypeptide or

polynucleotide. An activity may be, e.g., a catalytic activity, binding activity, ability to perform or participate in a biological function or process, etc. In some embodiments an activity is one that has (or the lack of which has) a detectable effect on an observable phenotype of a cell or organism. In some embodiments an activity of a variant may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, of the activity of the reference polypeptide or polynucleotide , up to approximately 100%, approximately 125%, or approximately 150% of the activity of the reference polypeptide or polynucleotide, in various embodiments . In some embodiments a variant, e.g., a functional variant, comprises or consists of a polypeptide at least 80%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%. 99.5% or 100% identical to an reference polypeptide or polynucleotide over at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 100% of the full length of the reference polypeptide or polynucleotide or over at least 70%, 75%, 80%, 85%, 90%, 92.5%, 95%, 96%, 97%, 98%, or 99% or 100% of a functional fragment of the reference polypeptide or polynucleotide. In some embodiments an alteration, e.g., a substitution or deletion, e.g., in a functional variant, does not alter or delete an amino acid or nucleotide that is known or predicted to be important for an activity, e.g., a known or predicted catalytic residue or residue involved in binding a substrate or cofactor. In some embodiments nucleotide (s) , amino acid(s), or region (s) exhibiting lower degrees of conservation across species as compared with other amino acids or regions may be selected for alteration. Variants may be tested in one or more suitable assays to assess activity. In certain embodiments a polypeptide or polynucleotide sequence in the NCBI RefSeq database may be used as a reference sequence. In some embodiments a variant or fragment of a naturally occurring polypeptide or

polynucleotide is a naturally occurring variant or fragment. In some embodiments a variant or fragment of a naturally occurring polypeptide or polynucleotide is not naturally occurring.

In some embodiments, the calcium-independent srtA mutant is selected according to the degree of sequence homology with a wild-type sortase A enzyme. In some embodiments , the calcium- independent srtA mutant is selected according to the degree of sequence homology with S. aureus sortase A. In some embodiments, the calcium- independent srtA mutant is selected according to the degree of sequence homology with SEQ ID NO: 1 (FIG. 4A) . In some embodiments, the calcium- independent srtA mutant is selected according to the degree of sequence homology with SEQ ID NO: 3 (FIG. 4C) . In some

embodiments, the calcium- independent srtA mutant is selected according to the degree of sequence homology with SEQ ID NO: 5 (FIG. 4E) . In some embodiments, the calcium- independent srtA mutant is selected according to the degree of sequence homology with SEQ ID NO: 7 (FIG. 4G) . Calcium- independent srtA mutants having a desired degree of homology to a wild-type sortase A enzyme can be identified by, e.g., using the wild-type sortase A nucleotide sequences as query sequences in a search against public databases to identify related sequences. For example, in some embodiments the calcium- independent srtA mutant comprises an amino acid sequence homologous to amino acids 60-206 of SEQ ID NO: 1, e.g. an amino acid sequence that is at least 55%, at least 60%, at least

65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or higher, homologous thereto. In some embodiments the calcium- independent srtA mutant comprises an amino acid sequence homologous to amino acids 60-206 of SEQ ID NO: 3, e.g. an amino acid sequence that is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or higher, homologous thereto. In some embodiments, a nucleotide sequence encoding a calcium- independent srtA mutant has at least 25%, or preferably at least 30%, or more preferably at least 35% or more identity with the nucleic acid sequence of SEQ ID NO : 2. In some embodimen s , a nucleotide sequence encoding a calcium- independent srtA mutant has at least 25%, or preferably at least 30%, or more preferably at least 35% or more identity with the nucleic acid sequence of SEQ ID NO: 4.

In further embodiments, the calcium- independent srtA mutant has at least 35%, or preferably at least 40%, or more preferably at least 45% similarity with the amino acid sequence of SEQ ID NO: 1. In further embodiments, the calcium- independent srtA mutant has at least 35%, or preferably at least 40%, or more preferably at least 45% similarity with the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the calcium-independent srtA mutant is a variant of SrtA of another Gram-positive bacterium having one or more as substitutions, deletions, insertions , and/or other modifications relative to the native nucleotide and/or amino acid sequence of S . aureus srtA. In some embodiments, the variant comprises one or more conservative amino acid substitutions relative to SrtA of another Gram-positive bacterium. In further embodiments , the variant comprises one or more amino acid substitutions relative to SrtA of another Gram-positive bacterium, wherein the one or more as amino acid

substitutions are predominantly, e.g., at least 50% , or preferably at least 60%, or more preferably at least 70% or more , conservative substitutions .

It will be appreciated that fragments of calcium- independent srtA mutants disclosed herein exhibiting calcium- independent sortase A catalytic activity are contemplated herein, and can be utilized in the methods described herein. As described in PCT/US2010/000274 , fragments can be identified by producing transaminase fragments by known recombinant techniques or proteolytic techniques , for example , and determining the rate of protein or peptide ligation. The fragment sometimes consists of about 80% of the full-length transamidase amino acid sequence, and sometimes about 70%, about 60%, about 50%, about 40% or about 30% of the full-length transamidase amino acid sequence such as that of a wild- type S. aureus Sortase A. In some embodiments, the fragment lacks an N-terminal portion of the full-length sequence, e.g., the fragment lacks the N-terminal portion extending to the end of the membrane anchor sequence. In some embodiments the fragment comprises the C-terminus of a full-length transamidase amino acid sequence. In some embodiments, a catalytic core region from a sortase is utilized, e.g., a region from about position 60 to about position 206 of SrtA, e.g., S. aureus SrtA. In some embodiments, the fragment comprises a sortase A lacking N-terminal amino acids 2-25 (SEQ ID NO: 5) (FIG. 4E) . An exemplary nucleotide sequence (i.e., SEQ ID NO: 6) encoding such fragment is shown in FIG. 4F.

Calcium-independent srtA mutants can be derived from sortase A sequences from other organisms. In some embodiments the disclosure provides mutants of any Ca⁺⁺- dependent SrtA from a species other than S. aureus, wherein the mutants comprise any of the mutations or combinations of mutations described herein at the corresponding positions in the Ca'^" -dependent SrtA from a species other than S. aureus. In some embodiments, a calcium- independent srtA mutant is derived from sortase A sequences from other organisms comprising nucleotide sequences substantially identical or similar to the nucleotide sequences that encode Srt A. A similar or substantially identical nucleotide sequence may include modifications to the native sequence, such as

substitutions, deletions, or insertions of one or more nucleotides. Included are nucleotide sequences that sometimes are about at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more identical to a native nucleotide sequence, and sometimes are about 90% or 95% or more identical to the native nucleotide sequence (each identity percentage can include a 1%, 2%, 3% or 4% variance) . One test for determining whether two nucleic acids are substantially identical is to determine the percentage of identical nucleotide sequences shared between the nucleic acids.

Calculations of sequence identity can be performed by a variety of techniques which are available to the skilled artisan. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., may be used to generate alignments and/or to obtain a percent identity.

In some embodiments, a calcium- independent srtA mutant comprises at least three amino acid substitutions relative to a wild-type sortase A, wherein the amino acid substitutions comprise a) a K residue at position 105; b) a Q or A residue at position 108; and c) at least one amino acid substitution selected from the group

consisting of i) a R residue at position 94; ii) a S residue at position 94; iii) a N residue at position 160; iv) a A residue at position 165; v) a E residue at position 190; and vi) a T residue at position 196. As used herein, "mutant" is used to refer to a one amino acid sequence which has changed by at least one amino acid residue relative to another amino acid sequence. The term "mutant" with reference to "sortases" should not be considered to imply that any particular way of generating the mutant sequences is required or that any particular starting materials is required . The present disclosure contemplates any suitable method of generating the calcium-independent srtA mutants described herein.

Examples of suitable methods include, but are not limited to introducing mutations into an appropriate wild-type coding sequence, synthesizing the sequences of the calcium-independent srtA mutants de novo, for example, utilizing solid phase peptide synthesis, and in vitro translation a synthetic mRNA, to name only a few. It is to be further understood that the disclosure contemplates calcium- independent mutants of any wild-type sortase A. Those skilled in the art will appreciate that the wild- type sequences of sortase A may vary, e.g., SrtA from various species may have gaps, insertions, and/or vary in length relative to the amino acid sequence of exemplary wild-type S. aureus SrtA. Accordingly, the disclosure is not intended to be limited in any way by the original amino acid residue at a particular position in any wild- type sortase A sequence used to generate a calcium- independent srtA mutant. Thus, any substitution which results in the specified amino acid residue at a position specified herein is contemplated by the disclosure. For the avoidance of doubt , the phrase "X residue at position Y" means that the X residue in the resulting mutant srtA replaces whatever amino acid was present in the original sortase A amino sequence at the position Y in the original sorta.se A amino acid sequence that corresponds to the same position in an exemplary wild-type S. aureus srtA amino acid sequence when accounting for any gaps and/or insertions in the original sortase A amino acid sequence relative to the exemplary wild-type S. aureus srtA amino acid sequence. The following examples are instructive and are not intended to be limiting in any way . If the original wild-type sortase A used to generate a srtA mutant is the wild-type S. pyogenes srtA (NCBI Gene ID: 901269), "position 94" in the phrase "an amino acid substitution comprising a R residue at position 94" would mean position 115 of the wild-type S. pyogenes srtA as position 115 of the wild-type S . pyogenes srtA corresponds to position 94 in the exemplary wild-type S. aureus srtA sequence (NCBI Gene ID: 3238307) when taking into account gaps and/or insertions in the sequence alignment of S. pyogenes srtA and S. aureus srtA. In such example, the N residue at position 115 of the wild- type S. pyogenes srtA amino acid sequence would be replaced by a R residue in the resulting mutant srtA.

If the original wild-type sortase A used to generate a srtA mutant is the wild-type B. anthracis srtA (NCBI Gene ID: 1088124), "position 94" in the phrase "an amino acid substitution comprising a R residue at position 94" would mean position 123 of the wild-type B . anthracis srtA as position 123 of the wild-type B . anthracis srtA corresponds to position 94 in the exemplary wild-type S. aureus srtA sequence (NCBI Gene ID: 3238307) when taking into account gaps and/or insertions .in the sequence alignment of B . anthracis srtA and S . aureus srtA. In such example, the E residue at position 123 of the wild- type B . anthracis srtA amino acid sequence would be replaced by a R residue in the resulting mutant srtA.

If the original wild-type sortase A used to generate a srtA mutant is the wild-type E. faecalis srtA (NCBI Gene ID: 1201902), "position 94" in the phrase "an amino acid substitution comprising a R residue at position 94" would mean position 112 of the wild- type E. faecalis srtA as position 112 of the wild- type E. faecalis srtA corresponds to position 94 in the exemplary wild- type S . aureus srtA sequence (Gene ID: 3238307) when taking into account gaps and/or insertions in the alignment of E. faecalis srtA and S . aureus srtA. In such example, the N residue at position 112 of the wild-type E. faecalis srtA amino acid sequence would be replaced by a R residue in the resulting mutant srtA.

Based on the teachings herein and examples above, those skilled in the art will understand how to align any original wild-type sortase A sequence to be used for generating a calcium- independent srtA mutant with an exemplary wild-type S. aureus sortase A sequence for purposes of determining the positions in the original wild-type sortase A sequence that correspond to the exemplary wild-type S . aureus sortase A sequence when taking into account gaps and/or insertions in the alignment of the two sequences.

In some embodiments, the substitution comprises a E105K substitution. It is to be understood that the phrase "E105K substitution", in reference to mutating a particular original sortase A sequence (e.g., a wild type sortase A sequence) refers to the substitution of a K residue at a position in the original sortase A amino acid sequence that corresponds to the E105 residue in the corresponding exemplary wild- type S. aureus srtA amino acid sequence . For example, if the original sortase A sequence to be mutated in accordance with the disclosure is a L. monocytogenes srtA sequence (NCBI Gene ID:

986837) , the phrase "E105K substitution" refers to the substitution of the R residue at position 112 of the L. monocytogenes srtA sequence wi h a K residue as position 112 of L. monocytogenes srtA corresponds to position 105 of the exem lary wild-type S. aureus srtA sequence (NCBI Gene ID : 3238307) when the two sequences are aligned taking into account any gaps and/or insertions. In some embodiments, the substitution comprises a E108Q

substitution. In some embodiments, the substitution comprises an E108A substitution. In some embodiments, the substitution comprises a P94R substitution. In some embodiments, the substitution comprises a P94S

substitution. In some embodiments, the substitution comprises a D160N substitution. In some embodiments, the substitution comprises a D165A substitution. In some embodiments, the substitution comprises a K190E

substitution. In some embodiments, the substitution comprises a K196T substitution.

In some embodiments, the wild-type sortase A comprises S. aureus sortase A. In some embodiments, the wild-type sortase A comprises an amino acid sequence of

SEQ ID NO: 1. In some embodiments, the wild-type sortase A comprises an amino acid sequence of SEQ ID NO: 3. In some embodiments, the calcium- independent srtA mutant comprises a deletion of amino acids 2-25. In some embodiments, the calcium- independent srtA mutant

comprises a deletion of amino acids 2-59.

In some embodiments , the calcium- independent srtA mutant comprises at least two amino acid substitutions selected from the group consisting of i) -vi) .

In some embodiments, the calcium-independent srtA mutant comprises at least two amino acid substitutions comprising i) and iii) . In some embodiments, the calcium-independent srtA mutant comprises at least two amino acid substitutions comprising i) and iv) . In some embodiments , the calcium-independent srtA mutant

comprises at least two amino acid substitutions

comprising i) and v) . In some embodiments , the calcium- independent srtA mutant comprises at least two amino acid substitutions comprising i) and vi) .

In some embodiments, the calcium-independent srtA mutant comprises at least two amino acid substitutions comprising ii) and iii) . In some embodiments, the calcium- independent srtA mutant comprises at least two amino acid substitutions comprising ii) and iv) . In some embodiments, the calcium- independent srtA mutant

comprises at least two amino acid substitutions

comprising ii) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least two amino acid substitutions comprising ii) and vi) .

In some embodiments, the calcium- independent srtA mutant comprises at least two amino acid substitutions comprising iii) and iv) . In some embodiments, the calcium- independent srtA mutant comprises at least two amino acid substitutions comprising iii) and v) . In some embodiments, the calcium- independent srtA mutant

comprises at least two amino acid substitutions

comprising iii) and vi) .

In some embodiments , the calcium- independent srtA mutant comprises at least two amino acid substitutions comprising iv) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least two amino acid substi utions comprising iv) and vi) .

In some embodiments , the calcium- independent srtA mutant comprises at least two amino acid substitutions comprising v) and vi) .

In some embodiments , the calcium- independent srtA mutant comprises at least three amino acid substitutions selected from the group consisting of i) -vi) .

In some embodiments , the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising i) , iii) and iv) . In some embodiments, the calcium-independent srtA mutant comprises at least three amino acid substitutions comprising i) , iii) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising i) , iii) and vi) .

In some embodiments, the calcium-independent srtA mutant comprises at least three amino acid substitutions comprising i) , iv) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising i) , iv) and vi) .

In some embodiments, the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising i) , v) and vi) .

In some embodiments, the calcium-independent srtA mutant comprises at least three amino acid substitutions comprising ii) , iii) and iv) . In some embodiments, the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising ii) , iii) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising ii) , iii) and vi) .

In some embodiments, the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising ii) , iv) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising ii) , iv) and vi) .

In some embodiments, the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising ii) , v) and vi) .

In some embodiments , the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising iii) , iv) and v) . In some embodiments , the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising iii), v) and vi) .

In some embodiments, the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising iv) , v) and vi) .

In some embodiments, the calcium- independent srtA mutant comprises at least four amino acid substitutions selected from the group consisting of i) -vi) .

In some embodiments, the calcium- independent srtA mutant comprises at least four amino acid substitutions comprising i) , iii), iv) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least four amino acid substitutions comprising i) , iii) , v) and vi) .

In some embodiments, the calcium- independent srtA mutant comprises at least four amino acid substitutions comprising ii) , iii) , iv) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least four amino acid substitutions comprising ii) , iii) , v) and vi) .

In some embodiments, the calcium- independent srtA mutant comprises at least four amino acid substitutions comprising iii) , iv) , v) and vi) .

In some embodiments, the calcium- independent srtA mutant comprises at least five amino acid substitutions selected from the group consisting of i) -vi) .

In some embodiments, the calcium- independent srtA mutant comprises at least five amino acid substitutions comprising i) , iii) , iv) , v) and vi) .

In some embodiments, the calcium- independent srtA mutant comprises at least five amino acid substitutions comprising ii), iii), iv), v) and vi) . In some embodiments, the calcium- independent srtA mutants comprises at least 60% identity to SEQ ID NO: 1, or at least 65% identity to SEQ ID NO: 1, or at least 70% identity to SEQ ID NO: 1, or at least 75% identity to SEQ ID NO: 1, or at least 80% identity to SEQ ID NO : 1, or at least 85% identity to SEQ ID NO: 1, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or up to 99% identity to SEQ ID NO: 1. In some embodiments, the calcium- independent srtA mutants comprises at least 60% identity to amino acid residues 60-206 of SEQ ID NO: 1, or at least 65% identity to amino acid residues 60-206 of SEQ ID NO: 1, or at least 70% identity to amino acid residues 60-206 of SEQ ID NO: 1, or at least 75% identity to amino acid residues 60-206 of SEQ ID NO: 1, or at least 80% identity to amino acid residues 60-206 of SEQ ID NO: 1, or at least 85% identity to amino acid residues 60-206 of SEQ ID NO: 1, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or up to 99% identity to amino acid residues 60-206 SEQ ID NO: 1.

In some embodiments, the calcium- independent srtA mutants comprises at least 60% identity to SEQ ID NO: 3, or at least 65% identity to SEQ ID NO: 3, or at least 70% identity to SEQ ID NO: 3, or at least 75% identity to SEQ ID NO: 3, or at least 80% identity to SEQ ID NO : 3, or at least 85% identity to SEQ ID NO: 3, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or up to 99% identity to SEQ ID NO: 3. In some embodiments, the calcium- independent srtA mutants comprises at least 60% identity to amino acid residues 60-206 of SEQ ID NO: 3, or at least 65% identity to amino acid residues 60-206 of SEQ ID NO: 3, or at least 70% identity to amino acid residues 60-206 of SEQ ID NO : 3, or at least 75% identity to amino acid residues 60-206 of SEQ ID NO: 3, or at least 80% identity to amino acid residues 60-206 of SEQ ID NO: 3, or at least 85% identity to amino acid residues 60-206 of SEQ ID NO: 3, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or up to 99% identity to amino acid residues 60-206 SEQ ID NO: 3.

In some embodiments, the calcium-independent srtA mutants comprises at least 60% identity to SEQ ID NO : 5, or at least 65% identity to SEQ ID NO: 5, or at least 70% identity to SEQ ID NO: 5, or at least 75% identity to SEQ ID NO: 5, or at least 80% identity to SEQ ID NO: 5, or at least 85% identity to SEQ ID NO: 5, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or up to 99% identity to SEQ ID NO: 5.

It should be appreciated that any of the calcium- independent srtA mutants disclosed herein can include a tag. In some embodiments, a calcium-independent srtA mutant comprises a C-terminal tag. In some embodiments, a calcium- independent srtA mutant comprises a N-terminal tag. In some embodiments, the calcium- independent srtA mutant comprises a N-terminal His6 tag. In some

embodiments , the calcium- independent srtA mutant

comprises a C- terminal His6 tag .

In some embodiments, the calcium- independent srtA mutants exhibit sortase A catalytic activity in the absence of calcium. In some embodiments, the calcium- independent srtA mutants exhibit sortase A catalytic activity in the absence of exogenous calcium.

In some embodiments, the calcium- independent srtA mutants exhibit sortase A catalytic activity in the presence of calcium-binding proteins.

In some embodiments, the calcium- independent srtA mutants exhibit sortase A catalytic activity in the presence of calcium concentrations up to 1 mM, up to 2 mM, up to 3 mM, up to 4 mM, up to 5 mM, up to 6 mM, up to 7 mM, up to 8 mM, up to 9 mM, or up to 10 mM or more.

In some embodiments, the calcium-independent srtA mutants exhibit sortase A catalytic activity in the presence of calcium concentrations less than 1 mM. In some embodiments, the calcium- independent srtA mutants exhibit sortase A catalytic activity in the presence of calcium concentrations less than 100 μΜ, less than 10 μΜ, less than 1 μΜ, less than 0.1 μΜ, or less than 0.01 μΜ.

In some embodiments, the calcium-independent srtA mutants exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least up to 80% of wild-type sortase A catalytic activity at 0 mM Ca²⁺ compared to wild-type sortase A catalytic activity at 10 mM Ca²⁺.

In some embodiments, the calcium-independent srtA mutants exhibit at least 85%, at least 90%, at least 91%, at least 92%, at least 90%, at least 91%, or at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of wild-type sortase A catalytic activity at 0 mM Ca²'^' compared to wild- type sortase A catalytic activity at 10 mM Ca²⁺.

In some embodiments, the calcium- independent srtA mutants exhibit at least 1- fold, at least 2 - fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least up to 100-fold, or more sortase A catalytic activity at 0 mM Ca²⁺ compared to wild-type sortase A catalytic activity at 10 mM Ca²⁺.

In some embodiments, the calcium- independent srtA mutants exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least up to 80% of sortase A catalytic activity of SEQ ID NO: 1 at 0 mM Ca²⁺ compared to sortase A catalytic activity of SEQ ID NO: 1 at 10 mM Ca²⁺ .

In some embodiments, the calcium- independent srtA mutants exhibit at least 85%, at least 90%, at least 91%, at least 92%, at least 90%, at least 91%, or at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of sortase A catalytic activity of SEQ ID NO: 1 at 0 mM Ca²⁺ compared to sortase A catalytic activity of SEQ ID NO: 1 at 10 mM Ca²" .

In some embodiments, the calcium- independent srtA mutants disclosed herein exhibit increased catalytic activity compared to the sortase A of SEQ ID NO: 1. In some embodiments, the calcium-independent srtA mutants exhibit at least 2-fold, at least 3-fold, at least 4- fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least up to 100- fold, or more than sortase A catalytic activity of SEQ ID NO: 1 at 0 mM Ca²⁺ compared to sortase A catalytic activity of SEQ ID NO: 1 at 10 mM Ca²⁺ .

In some embodiments, the calcium- independent srtA mutants exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least up to 80% of sortase A catalytic activity of SEQ ID NO: 3 at 0 mM Ca²⁺ compared to sortase A catalytic activity of SEQ ID NO: 3 at 10 mM Ca²⁺ .

In some embodiments, the calcium- independent srtA mutants exhibit at least 85%, at least 90%, at least 91%, at least 92%, at least 90%, at least 91%, or at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of sortase A catalytic activity of SEQ ID NO: 3 at 0 mM Ca²⁺ compared to sortase A catalytic activity of SEQ ID NO: 3 at 10 mM Ca²⁺ .

In some embodiments, the calcium- independent srtA mutants disclosed herein exhibit increased catalytic activity compared to the sortase A of SEQ ID NO: 3. In some embodiments, the calcium- independent srtA mutants exhibit at least at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold, at least 20-fold, at least 30-fold, at least 40- fold, at least 50-fold, at least 60-fold, at least 70- fold, at least 80-fold, at least 90-fold, or at least up to 100-fold, or more than sortase A catalytic activity of SEQ ID NO: 3 at 0 mM Ca²⁺ compared to sortase A catalytic activity of SEQ ID NO: 3 at 10 mM Ca²⁺ .

In some embodiments, the calcium- independent srtA mutants exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least up to 80% of sortase A catalytic activity of SEQ ID NO: 5 at 0 mM Ca²⁺ compared to sortase A catalytic activity of SEQ ID NO: 5 at 10 mM Ca²⁺.

In some embodiments, the calcium- independent srtA mutants exhibit at least 85%, at least 90%, at least 91%, at least 92%, at least 90%, at least 91%, or at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to

100% of sortase A catalytic activity of SEQ ID NO: 5 at 0 mM Ca²⁺ compared to sortase A catalytic activity of SEQ ID NO: 5 at 10 mM Ca²⁺ . In some embodiments, the calcium- independent srtA mutants disclosed herein exhibit increased catalytic activity compared to the sortase A of SEQ ID NO: 5. In some embodiments, the calcium- independent srtA mutants exhibit at least 2-fold, at least 3-fold, at least 4- fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least up to 100- fold, or more than sortase A catalytic activity of SEQ ID NO: 5 at 0 mM Ca²⁺ compared to sortase A catalytic activity of SEQ ID NO: 5 at 10 mM Ca²⁺ .

Aspects of the present disclosure also relate to calcium- independent mutants comprising an amino acid sequence at least 80% identical to SEQ ID NO: 9.

In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence at least 80% identical to SEQ ID NO : 9, wherein the mutant comprises a) a K residue at position 47 of SEQ ID NO: 9; b) a Q residue at position 50 of SEQ ID NO: 9; and c) at least one amino acid residue of SEQ ID NO: 9 selected from the group consisting of i) a R residue at position 36 of SEQ ID NO:9; ii) a N residue at position 102 of SEQ ID NO: 9; iii) a A residue at position 107 of SEQ ID NO: 9; iv) a E residue at position 132 of SEQ ID NO : 9; and v) a T residue at position 138 of SEQ ID NO : 9.

In some embodiments , a calcium- independent srtA mutant comprises an amino acid sequence at least 80% identical to SEQ ID NO : 9, wherein the mutant comprises a) a K residue at position 47 of SEQ ID NO : 9; b) a A residue at position 50 of SEQ ID NO: 9; and c) at least one amino acid residue of SEQ ID NO: 9 selected from the group consisting of i) a R residue at position 36 of SEQ ID NO: 9; ii) a N residue at position 102 of SEQ ID NO: 9; iii) a A residue at position 107 of SEQ ID NO: 9; iv) a E residue at position 132 of SEQ ID NO: 9; and v) a T residue at position 138 of SEQ ID NO: 9.

In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 16 (FIG. 4P) . In some embodiments, a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 16 comprising a A residue at position 50. In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 17 (FIG. 4Q) . In some embodiments, a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 17 comprising a A residue at position 50. In some

embodiments, a calcium-independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 18 (FIG. 4R) . In some embodiments, a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 18 comprising a A residue at position 50. In some

embodiments, a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO : 19 (FIG. 4S) . In some embodiments, a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 19 comprising a A residue at position 50. In some

embodiments, a calcium-independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 20 (FIG. 4T) . In some embodiments, a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 20 comprising a A residue at position 50. In some

embodiments, a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 21 (FIG. 4U) . In some embodiments, a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 21 comprising a A residue at position 50.

In some embodiments, a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) .

In some embodiments, a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising i) and ii) . In some embodiments, a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising i) and iii) . In some embodiments, a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising i) and iv) . In some embodiments, a calcium-independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising i) and v) .

In some embodiments, a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising ii) and iii) . In some embodiments, a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising ii) and iv) . In some embodiments , a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO : 9 comprising ii) and v) .

In some embodiments , a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO : 9 comprising iii) and iv) . In some embodiments , a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising iii) and v) .

In some embodiments , a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising iv) and v) . In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 15 (FIG. 40) . In some embodiments, a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 15 comprising a A residue at position 50.

In some embodiments, a calcium- independent srtA mutant comprises at least three amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) .

In some embodiments, a calcium- independent srtA mutant comprises at least three amino acid residues of SEQ ID NO: 9 comprising i) , ii) , and iii) . In some embodiments, a calcium- independent srtA mutant comprises at least three amino acid residues of SEQ ID NO: 9 comprising i) , ii) , and iv) . In some embodiments, a calcium- independent srtA mutant comprises at least three amino acid residues of SEQ ID NO: 9 comprising i) , ii) , and v) .

In some embodiments, a calcium- independent srtA mutant comprises at least three amino acid residues of SEQ ID NO: 9 comprising ii) , iii) , and iv) . In some embodiments, a calcium- independent srtA mutant comprises at least three amino acid residues of SEQ ID NO : 9 comprising ii) , iv) , and v) .

In some embodiments, a calcium- independent srtA mutant comprises at least three amino acid residues of SEQ ID NO : 9 comprising iii ) , iv) , and v) .

In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO : 12. (FIG . 4L) ) In some embodiments , a calcium- independent srtA mutant comprises an amino acid sequence SEQ ID NO:

13 (FIG . 4M) . In some embodiments , a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO : 14 (FIG . 4N) . In some embodiments , a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 14 comprising a A residue at position 50.

In some embodiments, a calcium- independent srtA mutant comprises at least four amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) .

In some embodiments, a calcium- independent srtA mutant comprises at least four amino acid residues of SEQ ID NO: 9 comprising i) , ii) , iii) , and iv) . In some embodiments, a calcium-independent srtA mutant comprises at least four amino acid residues of SEQ ID NO: 9 comprising i) , iii) , iv) , and v) . In some embodiments, a calcium- independent srtA mutant comprises at least four amino acid residues of SEQ ID NO: 9 comprising ii) , iii), iv) , and v) . In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 11 (FIG. 4K) . In some embodiments, a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 11 comprising a A residue at position 50.

In some embodiments, a calcium-independent srtA mutant comprises an amino acid sequence at least 90% identical to SEQ ID NO: 9. In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 9. In some embodiments, a calcium-independent srtA mutant comprises an amino acid sequence at least 96% identical to SEQ ID NO: 9. In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence at least 97% identical to SEQ ID NO: 9. In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence at least 98% identical to SEQ ID NO : 9. In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence at least 99% identical to SEQ ID NO : 9. In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 9.

It should be appreciated that with the amino acid sequences of the calcium-independent srtA mutants disclosed herein, the skilled person may determine suitable polynucleotides that encode calcium- independent srtA mutants. Accordingly, certain aspects of the disclosure provide polynucleotide sequences comprising the gene encoding calcium- independent srtA mutants, and their coding sequences. In some embodiments,

polynucleotides encoding mutants of sortase A comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 10 are disclosed. In some embodiments, a

polynucleotide encoding a mutant of sortase A (i.e., polynucleotides encoding calcium- independent srtA mutants) comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 10, and encodes a) a K residue at position 47 of SEQ ID NO: 9; b) a Q residue at position 50 of SEQ ID NO: 9; and c) at least one amino acid residue of SEQ ID NO: 9 selected from the group

consisting of i) a R residue at position 36; ii) a N residue at position 102; iii) a A residue at position 107; iv) a E residue at position 132; and v) a T residue at position 138.

In some embodiments, a polynucleotide encoding a mutant of sortase A (i.e., polynucleotides encoding calcium- independent srtA mutants) comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 10, and encodes a) a K residue at position 47 of SEQ ID NO: 9; b) a A residue at position 50 of SEQ ID NO: 9; and c) at least one amino acid residue of SEQ ID NO: 9 selected from the group consisting of i) a R residue at position 36; ii) a N residue at position 102; iii) a A residue at position 107; iv) a E residue at position 132; and v) a T residue at position 138.

In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) .

In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising i) and ii) . In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising i) and iii) . In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising i) and iv) . In some

embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising i) and v) .

In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising ii) and iii) . In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising ii) and iv) . In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising ii) and v) .

In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising iii) and iv) . In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising iii) and v) .

In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising iv) and v) .

In some embodiments, the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) . In some embodiments, the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9

comprising i) , ii) and iii) . In some embodiments, the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9 comprising i) , ii) and iv) . In some embodiments, the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9 comprising i) , ii) and v) .

In some embodiments, the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9

comprising ii) , iii) and iv) . In some embodiments, the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9 comprising ii) , iii) and v) .

comprising iii) , iv) and v) .

In some embodiments, the polynucleotide encodes at least four amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) .

In some embodiments, the polynucleotide encodes at least four amino acid residues of SEQ ID NO: 9 comprising i) , ii) , iii) , and iv) . In some embodiments , the polynucleotide encodes at least four amino acid residues of SEQ ID NO: 9 comprising ii) , iii) , iv) and v) .

In some embodiments, the polynucleotide encodes at each of the amino acid residues of SEQ ID NO: 9.

In some embodiments, a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 85% identical to SEQ ID NO: 10. In some embodiments, a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 10. In some embodiments, a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 96% identical to SEQ ID NO: 10. In some embodiments, a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 97% identical to SEQ ID NO: 10. In some embodiments, polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 98% identical to SEQ ID NO: 10. In some embodiments, a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 99% identical to SEQ ID NO: 10. In some embodiments, a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 100% identical to SEQ ID NO: 10. In some embodiments, a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence of SEQ ID NO: 10.

Certain aspects of the disclosure relate to a nucleic acid construct comprising a polynucleotide disclosed herein (e.g., a polynucleotide encoding a calcium- independent srtA mutant) .

In some embodiments, the nucleic acid construct comprises a nucleotide sequence that encodes one or more C-terminal or N-terminal tags.

The term "polynucleotide" refers to a molecule, which is a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule, either single stranded or double stranded. The polynucleotides of the present disclosure, such as polynucleotides encoding the calcium- independent srtA mutants, can be isolated or synthesized using standard molecular biology techniques and the sequence information provided herein. The synthetic

polynucleotides may be optimized in codon use, preferably according to the methods described in WO2006/077258 and/or PCT/EP2007/055943 , which are herein incorporated by reference. PCT/EP2007/055943 addresses codon-pair optimization. The polynucleotides encoding the calcium- independent srtA mutants of the disclosure can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PGR amplification techniques. The nucleic acid so amplified can be cloned into an

appropriate vector and characterized by DNA sequence analysis. A polynucleotide may either be present in isolated form, or be comprised in recombinant nucleic acid molecules or vectors, or be comprised in a host cell. Herein standard isolation, hybridization,

transformation and cloning techniques are used (e.g., as described in Sambrook, J., Fritsh, E. F . , and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed . , Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

By "isolated" polypeptide or protein is intended a polypeptide or protein removed from its native

environment. For example, recombinant.ly produced

polypeptides and proteins expressed in host cells are considered isolated for the purpose of the disclosure, as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single- step purification method

disclosed in Smith and Johnson, Gene 67:31-40 (1988) . As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

Expression vectors useful in the present disclosure include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids,

bacteriophage, yeast episome , yeast chromosomal elements, viruses such as baculoviruses , papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids . Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms

"transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic

lipidmediated transfection or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al . (Molecular Cloning: A

Laboratory Manual, 2^nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), Davis et al . , Basic Methods in Molecular Biology (1986) and other laboratory manuals .

It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be

transformed, the level of expression of protein desired, etc. The vectors, such as expression vectors, of the disclosure can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein (e.g. calcium- independent srtA mutant proteins, catalytically active fragments, catalytically active variants or catalytically active derivatives thereof) . The vectors, such as recombinant expression vectors, of the disclosure can be designed for expression of calcium-independent srtA mutant proteins in

prokaryotic or eukaryotic cells.

For example, calcium- independent srtA mutant proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression

vectors), filamentous fungi, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in

Enzymology 185, Academic Press, San Diego, Calif. (1990) . Representative examples of appropriate hosts are

described hereafter.

A "host cell," as used herein, is any cell capable of being grown and maintained in cell culture under conditions allowing for production and recovery of useful quantities of a biological product, as defined herein. Host cells can be unmodified cells or cell lines, or cell lines which have been genetically modified (e.g., to facilitate production of a biological product) . In some embodiments, the host cell is a cell line that has been modified to allow for growth under desired conditions, such as in serum-free media, in cell suspension culture, or in adherent cell culture.

A host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion . Such modifications (e.g. , glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein. Suitable host cells are preferably prokaryotic microorganisms such as bacteria (e.g., E. coli) , or in some embodiments eukaryotic organisms, for example f ngi, such as yeasts or filamentous fungi, or plant cells.

The calcium- independent srtA mutant according to the disclosure can be recovered and purified from recombinant cell cultures by methods known in the art (e.g., ion- exchange chromatography, hydrophobic interaction

chromatography, affinity chromatography and size

exclusion chromatography to further separate the target calcium- independent mutant srtA from the bulk protein to enable recovery of the target calcium- independent srtA mutant in a highly purified state) .

In some aspects the disclosure relates to a method of producing a calcium- independent srtA mutant comprising the steps of: (a) culturing the host cell according to the disclosure in a suitable culture medium under suitable conditions to produce calcium- independent srtA mutant; and optionally (b) purifying said calcium- independent srtA mutant to provide a purified calcium- independent srtA mutant product .

Another aspect of the present disclosure relates to methods for the production of substantially purified calcium- independent srtA mutant enzyme of the present disclosure. It should be appreciated that such enzyme can be prepared in compliance with Good Manufacturing Practices (GMP) or used in a GMP-com liant process.

An exemplary method for the production of

substantially purified calcium- independent srtA mutant involves cloning a nucleic acid segment encoding the calcium- independent srtA mutant enzyme can be inserted in a vector that contains sequences allowing expression of a sortase-transamidase in another organism, such as E.

coli. A suitable host organism can then be transformed or transfected with the vector containing the cloned nucleic acid segment. Expression is then performed in that host organism. The expressed enzyme is then purified using standard techniques. Techniques for the purification of cloned proteins are well known in the art and need not be detailed further here (e.g., affinity chromatography on a nickel NTA column for purification of a calcium- independent srtA mutant enzyme extended at its carboxyl terminus with a sufficient number of histidine residues to allow specific binding of the protein molecule to the nickel NTA column through the histidine residues) .

In some aspects the disclosure further provides an enzyme composition comprising one or more calcium- independent srtA mutants. In some aspects the disclosure relates to a sortase-mediated transpeptidation reaction catalyzed by an enzyme composition according to the disclosure. Examples of synthetic nucleophiles that can be used in such sortase-mediated transpeptidation reactions are shown in Table 1 below .

Table 1: Examples of synthetic nucleophiles used in site-specific sortase A transpeptidation reactions .

Probe ^La] Labeling Property Endowed

Site

H-G5K(biotin) L-OH C Term Biophysical handle

H-G5K (A P) K (Biotin) I -OH C Term Biophysical

handle/photocleavage

H-G5K (phenylazide) K (biotin) C Term Biophysical

G-OH handle/photo-crosslinker

H-G3K(FITC) -NH2 C Term Fluorescence

H-G3K (K (TAMRA) ) -NH2 C Term Fluorescence

H-G3YC (biotin) -NH2 C Term Biophysical handle

H-G3YC (Alexa 488) -NH2 C Term Fluorescence

H-AA-Ahx-K (K (TAMRA) ) -NH2 C Term Fluorescence

(S.

pyogenes)

H-G3K(C12-C24) -NH2 C Term Lipidation

H-G3K(l-ad) -NH2 C Term Hydrophobicity

H-G3WK (cholesterol) -NH2 C Term Lipidation

d-Tat (1^st residue is G) C Term Cell Penetration

H-G2Y-PTD5-NH2 C Term Cell Penetration

(H2NRRQRRTSKLMKRAhx) 2KYK (GG C Term Cell Penetration

-NH2) -NH2

H-G3K(folate) -NH2 C Term Folic Acid

H2N-PEG G Term Inert Polymer

H-G3K (PEG) -OH C Term Inert Polymer

H-G3 -MAP-NH2 C Term Cell Penetration

Aminoglycoside antibiotics C Term Antibiotic

(various)

C Term GPI Mimic

GPI mimics based on 19 with C Term GPI Mimic

trisaccharide cores

Biotin-PEG-YGLPETGG-NH2 N Term Biophysical handle

Alexa 64 / -LPETGG-NH2 N Term Fluorescence

Alexa 488 -LPETGG-NH2 N Term Fluorescence

Biotin-LPRT-OMe N Term Biophysical handle

FITC-Ahx-LPRT-OMe N Term Fluorescence

FAM-LPETG-NH2 N Term Fluorescence

Biotin-GGLPETG-NH2 N Term Biophysical handle

N3-ALPETG-NH2 N Term Handle for Bioorthognal chmical reactions [a] 1-Ad = 1-adamantyl, Ahx=aminohexanoic acid, FAM=carboxyfluorescein, FITC = fluorescin isothiocyanate , PEG = polyethylene glycol, TAMRA =

carboxytetramethylrhodamine

In some aspects the disclosure relates to the use of an enzyme composition disclosed herein for the sortagging of a protein. An enzyme composition of the disclosure may comprise a polypeptide which has the same enzymatic activity, for example the same type of transamidase activity as that provided by a polypeptide of the disclosure. An enzyme composition of the disclosure may comprise a polypeptide which has a different type of enzymatic activity than that provided by a polypeptide of the disclosure. In some embodiments, the enzyme

composition is purified to comprise calcium- independent srtA mutants of a particular sequence (e.g., SEQ ID NO: 9) . In some embodiments, the enzyme composition

comprises calcium- independent srtA mutants predominantly in monomeric form. In some embodiments, the enzyme composition comprises a mixture of both monomeric and dimeric calcium- independent srtA mutants . In some embodiments , the enzyme composition comprises a mixture of calcium- independent srtA mutants comprising different sequences. In some embodiments, the enzyme composition comprises at least two, at least three , at least four, or at least five calcium- independent sortase A mutants selected from the group consisting of SEQ ID NO : 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13 , SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 , SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , and combinations thereof . In some embodiments , the enzyme composition comprises a group of calcium- independent sortase A mutants comprising SEQ ID NO : 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:

13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID

NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO : 20, SEQ ID NO: 21, and combinations thereof. In some

5 embodiments, the enzyme composition comprises a calcium- independent srtA mutant and a calcium-dependent srtA

mutant. In some embodiments, the enzyme composition

comprises a calcium- independent srtA mutant of SEQ ID NO: 9 and a calcium-dependent srtA mutant of SEQ ID NO: 7.

10 In some embodiments, the enzyme composition comprises a

wild-type srtA enzyme and a calcium- independent srtA

mutant. In some embodiments, the enzyme composition

comprises a calcium- independent srtA mutant of SEQ ID NO:

9, and a wild-type srtA enzyme of SEQ ID NO: 1, SEQ ID

15 NO: 3 or SEQ ID NO: 5. In some embodiments, the enzyme

composition comprises an aqueous medium.

The calcium- independent srtA mutants and enzyme compositions comprising the calcium- independent srtA

mutants disclosed herein can be used in any application

20 for which sortase-mediated ligation is desirable.

Exemplary such applications include, but are not limited to , specific incorporation of novel functionality into proteins , synthesis of neoglycoconjugates , immobilization of proteins to solid surfaces, protein labeling on living

25 cells, and protein circularization/dimerization, as is

shown in Table 2 below.

Table 2 Summary of applications for sortase-mediated ligation

Target Proteins Molecular Probe References

( 1) Specific incorporation of novel functionality into proteins

GFP-LPETG-Hisg Folate, (D)-Tat, AT-P-022, G2Y- Mao (2004)

PTD5, Gly-emGFP GFP-LPETG-His₆ Polyethyleneglycol (PEG) Parthasart hy et al . (2007)

H-2K^b-LPETG Biotin, phenylazide Popp et photocrosslinker, FITC, al. (2007) tetramethylrhodamine (TAMRA) 3- amino-3 - (o-nitrophenyl) ropionic

acid

CXCL14 -LPETG TAMRA Popp et al. (2007)

GFP-LPETG Fatty acids, cholesterol Antos et al. (2008)

PNA-LPKTGG Amphipathic peptide (MAP) Pritz et al. (2007)

GBl-VcSH3-LPETG-His₅ Unlabelled GBl for NMR analysis Kobashigaw a et al . (2009)

(2) Synthesis of neoglycoconj ugates

YALPETGK peptide 6 -deoxy- 6 -amino-glucose, Samantaray

6 -deoxy- 6 -amino-mannose et al .

(2008)

Rev-LPETGK Kanamycin, ribostamycin, neomycin Samantaray et al .

(2008)

Tat -LPETGK Kanamycin, ribostamycin, neomycin Samantaray et al . (2008)

Mrp-LPNTG-X_n Tobramycin Samantaray et al . (2008)

Target Proteins Surface References

(3) Immobilisation of proteins to solid surfaces

eGFP-LPETG Polystyrene beads Parthasart hy et al . (2007) eGFP-LPETGG-His_f, Glycidyl methacrylate (GMA) Chan et al. (2007)

Tus-LPETGG-His₆ Glycidyl methacrylate (GMA) Chan et al. (2007)

Fbp-LPETG C 5 biosensor chip Chow et al. (2008) Target Proteins Molecular Probe References

(4) Protein labeling on living cell

CD154 -LPETG Biotin, TAMRA Popp et al. (2007)

Neuraminidase-LPETG Biotin Popp et al. (2007)

ODF-LPETGG Alexa Fluor 488, biotin T¾n¾-ki3. St

al. (2008)

LPETGs-ECFP LPETGG-Alexa Fluor 647, LPETGG- Yamamoto biotin et al .

(2009)

(5) Protein circularisation

Glyv,-EGFP-LPETG-His_s - - - Antos et al. (2009)

The calcium- independent srtA mutants according to the disclosure may feature a number of significant

advantages over existing srtA variants currently used.

Depending on the specific application, these advantages may include aspects such as lower production costs,

higher specificity towards the substrate, reduced

antigenicity, fewer undesirable side activities, higher yields when produced in a suitable microorganism, more suitable pH and temperature ranges, calcium- independence, i.e., the ability to exhibit sortase A catalytic activity in environments in which calcium is low or unavailable , such as the cytoplasm, or in the presence of calcium- binding proteins, or when calcium-binding substances such as phosphate , carbonate, and EDTA are present . In

various embodiments, a calcium- independent srtA mutant or composition of the disclosure may be used in any process which requires the sortagging of a moiety of interest to a target protein . The terms "sortagging" , "sortase- mediated ligation", "sortase-mediated transpeptidation" , "sortase-mediated transacylation" , are used

interchangeably herein to refer to the process of adding a tag. It should be appreciated that any tag can be used. Examples of suitable tags include, but are not limited to, amino acids, peptides, proteins, nucleic acids, polynucleotides, sugars, carbohydrates, polymers, lipids, fatty acids, and small molecules. Other suitable tags will be apparent to those of skill in the art and the disclosure is not limited in this aspect.

In some embodiments, a tag comprises a sequence useful for purifying, expressing, solubilizing, and/or detecting a polypeptide. In some embodiments, a tag comprises an HA, TAP, Myc, 6XHis, Flag, or GST tag, to name few examples . In some embodiments a tag comprises a solubility-enhancing tag (e.g., a SUMO tag, NUS A tag, SNUT tag, a Strep tag, or a monomeric mutant of the Ocr protein of bacteriophage T7) . See, e.g., Esposito D and Chatterjee DK. Curr Opin Biotechnol . ; 17(4):353-8 (2006). In some embodiments, a tag is cleavable, so that it can be removed, e.g., by a protease. In some embodiments, this is achieved by including a protease cleavage site in the tag, e.g., adjacent or linked to a functional portion of the tag. Exemplary proteases include, e.g., thrombin, TEV protease, Factor Xa, PreScission protease, etc. In some embodiments, a "self-cleaving" tag is used. See, e.g., PCT/US05/05763.

In some embodiments, a tag comprises a click chemistry handle.

In some embodiments, the calcium-independent srtA mutants disclosed herein can be used to sortag cells to be administered to a subject, e.g., human subjects. In some embodiments, a method for sortagging a target polypeptide comprises (a) providing a polypeptide comprising a sortase recognition motif; (b) providing a moiety comprising an N-terminal oligoglycine sequence or a terminal alkylamine; and (c) contacting the target polypeptide with the moiety in the presence of an enzyme composition described herein (e.g., a composition comprising at least one calcium- independent srtA mutant disclosed herein) under conditions suitable for the srtA A mutant to ligate the moiety to the target protein, thereby sortagging the target protein.

It should be appreciated that the sortagging methods disclosed herein can be used to attach a moiety to any target protein or polypeptide. Methods and compositions provided herein can be used to conjugate essentially any polypeptide to any moiety. Non- limiting examples of polypeptides that can be produced or conjugated according to methods provided herein include receptors, membrane proteins, cytokines, chemokines, hormones, enzymes, growth factors, growth factor receptors, antibodies, antibody derivatives and other immune effectors,

interleukins , interferons, erythropoietin, integrins, soluble major histocompatibility complex antigens, binding proteins, transcription factors, translation factors, oncoproteins or proto-oncoproteins , muscle proteins, myeloproteins, neuroactive proteins, tumor growth suppressors, structural proteins, and blood proteins (e.g., thrombin, serum albumin, Factor VII, Factor VIII, Factor IX, Factor X, Protein C, von

Willebrand factor, etc.). In some embodiments, the polypeptide is a glycoprotein or other polypeptide which requires post- translational modification, such as deamidation, glycation, or the like, for optimal activity. In some embodiments, the polypeptide is a lipoprotein. Exemplary target polypeptides which have been sortagged are shown in Table 3 below.

Table 3 : Examples of proteins labeled by sortase A transpeptidation .

[a] The numbers denote the probe identities from Table 1

In some embodiments the target polypeptide comprises or consists of a polypeptide that is at least 80%, or at least 90%, e.g., at least 95%, 86%, 97%, 98%, 99%, 99.5%,

Substrate Solution/Cel Labeling Label (s) ^LaJ

1 Surface

H-2K^b Solution C Terminal __L 2 3 1

CD154 Cell surface C Terminal 1,5 neuraminidase Cell surface C Terminal 1

ODF Cell surface C Terminal 6,7

CRE Solution C Terminal 5

UCHL3 Solution C Terminal 1

P97 Solution C Terminal 5 eGFP Solution C Terminal 9, 10, 11

GFP Solution C Terminal 13 , 14, 15

PNA Solution C Terminal 18 eGFP Solution C Terminal 16, 17

Mrp Solution C Terminal 19

YALPETGK Solution C Terminal 19

(His) 6YALPETGKS Solution C Terminal 20

CD52 peptides Solution C Terminal 21

CD24 Solution C Terminal 21

MUC1 Solution C Terminal 21

LPETG5 -ECFP-TM Cell surface N Terminal 22, 23

LPETG5-PAFR Cell surface N Terminal 24

G3 - /G5-CTXB Solution N Terminal 25,26

G3 -eGFP Solution N Terminal 26

G-UCHL3 Solution N Terminal 26

S . aureus Cell surface N Terminal 27,28,29 surface

peptidoglycan

eGFP Solution N and C 26 and 8

Terminal

UCHL3 Solution N and C 26 and 8

Terminal or 100% identical to a naturally occurring protein or polypeptide. In some embodiments, the target polypeptide has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences relative to a naturally occurring sequence. In some embodiments the naturally occurring protein is a mammalian protein, e.g., of human origin. Naturally occurring sequences, e.g., genomic, mRNA, and polypeptide sequences, from a wide variety of species, including human, are known in the art and are available in publicly accessible databases such as those available at the National Center for Biotechnology Information (www.ncbi.nih.gov) or Universal Protein Resource

(www.uniprot.org) . Databases include, e.g., GenBank, RefSeq, Gene, UniProtKB/SwissProt , UniProtKB/Trembl , and the like. Sequences, e.g., nucleic acid (e.g., mRNA) and polypeptide sequences, in the NCBI Reference Sequence database may be used as reference sequences.

In some embodiments, a target polypeptide is a protein that is approved by the US Food & Drug

Administration (or an equivalent regulatory authority such as the European Medicines Evaluation Agency) for use in treating a disease or disorder in humans . Such proteins may or may not be one for which a PEGylated version has been tested in clinical trials and/or has been approved for marketing .

In some embodiments , a target polypeptide is a neurotrophic factor, i.e., a factor that promotes survival , development and/or function of neural lineage cells (which term as used herein includes neural

progenitor cells , neurons , and glial cells , e.g., astrocytes, oligodendrocytes, microglia) .

In some embodiments , t e target protein is one that forms homodimers or heterodimers , (or homo- or heterooligomers comprising more than two subunits, such as tetramers) .

In some embodiments, the target polypeptide is an enzyme, e.g., an enzyme that is important in metabolism or other physiological processes. In some embodiments, a target protein comprises a receptor or receptor fragment (e.g., extracellular domain).

In some embodiments, the target polypeptide is sortagged in cells to be administered to a subject. The present disclosure contemplates any application for which administration of cells comprising a sortagged

polypeptide is desirable (e.g., therapeutic, diagnostic, imaging, etc.) As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal.

Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice , rats, woodchucks , ferrets , rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species , e.g., chicken, emu, ostrich, and fish, e.g., trout , catfish and salmon . Subj ect includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans , primates or rodents . In certain embodiments, the subj ect is a mammal, e.g., a primate, e.g., a human . Preferably, the subj ect is a mammal . The mammal can be a human, non- human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used, for example, as subj ects that represent animal models of disease . A subj ect can be male or female . One of skill in the art will be aware that certain proteins, e.g., secreted eukaryotic (e.g., mammalian) proteins, often undergo intracellular processing (e.g., cleavage of a secretion signal prior to secretion and/or removal of other portion (s) that are not required for biological activity) , to generate a mature form. Such mature, biologically active versions of target proteins are used in certain embodiments of the disclosure.

A "moiety" to be conjugated (ligated) to a

polypeptide according to methods provided herein can be any agent suitable for conjugation to a polypeptide, i.e., capable of being operably linked to a sortase recognition sequence. The moiety can confer any of a number of possible functionalities to the polypeptide, such as but not limited to, altered physico-chemical properties, such as solubility and/or stability,- altered pharmacokinetic properties, such as bioavailability, clearance rate, and/or plasma half-life and/or altered biological activity, such as immunogenicity and/or antigenicity. Non-limiting examples of moieties include: a small -molecule , a peptide, a polypeptide, a lipid and/or fatty acid, a carbohydrate, a nucleic acid, a reporter molecule (e.g., a reporter enzyme, fluorescent molecule, a radiolabel, an affinity label, or the like), a toxin, a therapeutic agent, a nanoparticle, a resin, a cell, a virus particle, an adjuvant molecule, or a polymer (e.g., a hydrophilic polymer), an affinity tag (e.g., His6) , or the like. In one embodiment, the moiety is a pharmacological carrier molecule.

In some embodiments, the moiety comprises, consists essentially of, or consists of a member of a prosthetic binding group, such as biotin/avidin,

biotin/streptavidin, maltose binding protein/maltose, glutathione S-transferase/glutathione ,

metal/polyhistidine, antibody/epitope , antibody/antigen, antibody/protein A or protein G, hapten/anti-hapten, folic acid/folate binding protein, vitamin B 12/intrinsic factor, nucleic acid/complementary nucleic acid,

sulfhydryl/maleimide, sulfhydryl/haloacetyl derivative, amine/isotriocyanate , amine/succinimidyl ester, or amine/sulfonyl halides .

In some embodiments, the moiety comprises, consists essentially of, or consists of a peptide, a

peptidomimetic (e.g., a peptoid) , an amino acid, an amino acid analog, a polynucleotide or polynucleotide analog, a nucleotide or nucleotide analog, or an organic or inorganic compound having a molecular weight between about 500 and about 10,000.

In some embodiments, the moiety comprises, consists essentially of, or consists of a second polypeptide. The polypeptide can be any polypeptide. For example, a protein which is difficult to produce in a cell (e.g., either due to toxicity) , can be expressed as two

fragments which can be j oined using the methods described herein (e.g. , first portion of the protein can be attached to the second portion, reconstituting an active protein using the methods described herein) .

In some embodiments , the moiety comprises , consists essentially of , or consists of a reporter molecule , such as a fluorescent molecule (e.g. , umbelliferone ,

fluorescein, fluorescein isothiocyanate , rhodamine , dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin) ; a radioisotope (e.g., Cu-64, Ga67, Ga-68, Zr-89, Ru-97, Tc-99, Rh-105, Pd-109, In- 1.1.1, 1-123 , I- 125, 1-131, Re-186, Re- 188, Au-198 , Pb-203 , At-211, Pb- 212 or Bi-212) a detectable enzyme (e.g. , horseradish peroxidase, alkaline phosphatase, p-galactosidase, or acetylcholinesterase); a luminescent material (e.g., luminol) ; or a bioluminescent material (e.g., luciferase, luciferin, or aequorin) .

In some embodiments, the moiety comprises, consists essentially of, or consists of a biologically active molecule, such as a toxin (e.g., abrin, ricin A,

pseudomonas exotoxin or diphtheria toxin) .

In some embodiments, the moiety comprises the polypeptide itself, such that the polypeptide is cyclized by the conjugation. Advantageously, cyclized proteins often exhibit desired properties relative to the

corresponding linear protein, such as enhanced

solubility, enhanced stability, enhanced plasma half-life and/or decreasing immunogenicity . In other embodiments, the polypeptide can be 'chained' (e.g., dimerized, trimerized, etc) .

In some embodiments, the moiety is a water-soluble polymer, non-peptidic polymer with an average molecular weight of about 200 to about 200,000 Daltons, depending on the desired effect on the properties of the

polypeptide. For example, in some embodiments, the moiety comprises, consists essentially of, or consists of a polymeric group, such as polyalkylene oxide (PAO) , polyalkylene glycol (PAG) , polyethylene glycol (PEG) , methoxypolyethylene glycol (mPEG) , polypropylene glycol (PPG), branched PEGs, copolymers of ethylene glycol and propylene glycol, polyvinyl alcohol (PVA) ,

polycarboxylate, poly-vinylpyrrolidone , polyethylene-co- maleic acid anhydride, polystyrene-co-maleic acid anhydride, dext rboxymethy1 -dextran,

polyoxyethylated glycerol, polyoxyethylated sorbitol, polyoxyethylated glucose, dextran, polyoxazoline, polyacryloylmorp oline, or a serum protein binding- ligand, such as a compound which binds to albumin (e.g., fatty acids, C₅-C₂4 fatty acid, aliphatic diacid (e.g. C₅- C₂₄) ) . Additional polymers useful in methods and

compositions provided herein are known in the art and are described, e.g., in U.S. Pat. No. 5,629,384, which is herein incorporated by reference.

In some aspects, the target polypeptide or moiety comprises an affinity tag that can be used to facilitate recovery and/or isolation of the conjugated polypeptide.

An affinity tag used in a method or composition provided herein can comprise any peptide or other molecule for which an antibody or other specific binding agent is available. Affinity tags known in the art as being useful for protein purification include, but are not limited to, a poly-histidine segment, protein A (e.g., Nilsson et al . , EMBO J. 4:1075 (1985); Nilsson et al . , Methods Enzymol . 198:3 (1991)), glutathione S transferase (e.g., Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (e.g., Grussenmeyer et al . , Proc . Natl. Acad. Sci. USA 82:7952 (1985)), substance P, FLAG peptide (e.g., Hopp et al . , Biotechnology 6:1204 (1988)), c-myc tags (detected with anti-myc antibodies) ,

calmodulin binding protein, and streptavidin binding peptide.

In some embodiments, an affinity tag described herein allows for selective enrichment of desired conjugation products. In some embodiments, an affinity tag is located N- terminal of a sortase recognition sequence or C- terminal of an oligoglycine sequence so that the tag remains associated with the polypeptide after sortase- catalyzed cleavage and ligation. As such, the affinity tag is retained in the conjugated polypeptide upon cleavage and/or ligation of the sortase recognition sequence by a sortase and affinity

purification isolates the intact conjugated polypeptide.

In some embodiments, an affinity tag is located C- terminal of a sortase recognition sequence so that the tag is cleaved from the polypeptide upon sortase- catalyzed cleavage and ligation. For example, the affinity tag is located C-terminal of the sortase recognition sequence (i.e., the sortase recognition sequence is between the target polypeptide and the affinity tag) . As such, after performing the cleavage and ligation reactions, the affinity tags are no longer attached to the target polypeptide, and fragments containing the affinity tag are easily removed by affinity purification.

In some aspects, the target polypeptide comprises a spacer peptide. For example, in some embodiments, a spacer peptide separates the target polypeptide from a sortase recognition sequence and/or an affinity tag, and/or the sortase recognition sequence from an affinity tag. A spacer peptide can be of any size, e.g., from several to 30 or more amino acid residues, sufficient to serve the intended purpose. Spacer peptides can enhance conformational flexibility between two or more domains of a protein and/or minimize steric interference with the folding and/or function of two or more domains of a protein. A spacer peptide will generally comprise an inert, flexible amino acid sequence, e.g., comprising predominantly glycine, serine, and/or alanine residues. In some embodiments, a spacer peptide sequence can be modified with one or more proline residues at the beginning and/or at the end of the spacer in order to isolate the spacer as a separate functional domain from neighboring domains of the protein. A variety of spacer peptides are known in the art.

"Contacting" according to methods provided herein refers to the addition of the target polypeptide to the culture medium in a manner that allows the calcium- independent srtA mutant to ligate the moiety to target polypeptide. In some embodiments, the contacting includes culturing the cells for a defined period of time in the presence of the target polypeptide. In other embodiments, the contacting includes culturing the cells for a variable period of time until a desired endpoint or other indicator is achieved.

Conditions which allow the calcium- independent srtA mutant to cleave the sortase recognition sequence and ligate the moiety to the target polypeptide, include for example, standard cell growth conditions known to those of skill in the art, e.g. for mammalian cells; 37° C . , 5% C0₂, and an appropriate cell culture medium. The cell culture medium may vary depending upon the host cell and can be determined readily by those of skill in the art. In some embodiments, the "contacting" step in the method takes place in the absence of calcium, i.e., there is no exogenous calcium added to the culture medium or reaction mixture .

In some embodiments a sorta.se is immobilized by attaching it to a support. As used herein, a "support" may be any entity or plurality of entities having a surface to which a substance may be attached or on which a substance may be placed. Examples, include, e.g., particles, slides, filters, interior wall or bottom of a vessel (e.g., a culture vessel such as a plate or flask, well of a microwell plate, tube), chips, etc. A support may be composed, e.g., of glass, metal, gels (e.g., agarose) , ceramics, polymers, or combinations thereof.

Immobilization may comprise contacting sortase or a composition containing sortase with an affinity reagent, e.g., an antibody, that binds to sortase, wherein the affinity reagent is attached to a support. In some embodiments the sortase is tagged, and the affinity reagent binds to the tag. In some embodiments sortase may comprise a tag, e.g., a 6X-His tag, which may be used to immobilize the sortase to a metal-ion containing resin or substrate. In some embodiments sortase is immobilized to magnetic particles. It will be understood that magnetic particles may be magneti sable and paramagnetic, e.g., superparamagnetic, i.e., they may only magnetic in a magnetic field. In some embodiments the support is in a column. Unreacted sortase substrates and reaction products may readily be separated from an immobilized sortase.

It will be understood that in some aspects, the disclosure encompasses agents produced according to methods described herein, and compositions comprising such agents. It will be understood that, in some aspects, the disclosure encompasses methods of using such agents, e.g., for one or more purposes described herein.

The disclosure further provides packaged products and kits, including calcium- independent srtA mutants described herein or polynucleotides encoding the same, cell lines, cell cultures, populations and compositions, including, as well cells, cultures, populations, and compositions enriched or selected for any calcium- independent srtA mutants or variants thereof, packaged into suitable packaging material. In some embodiments, a packaged product or kit includes calcium- independent srtA mutants in monomeric form. In some embodiments, the packaged product or kit includes calcium-independent srtA mutants in dimeric form. In some embodiments, the packaged product or kit includes a mixed population of monomeric and dimeric calcium- independent srtA mutants.

In some aspects, a packaged product or kit includes a label, such as a list of the contents of the package, or instructions for using the kit e.g., instructions for sortagging a target polypeptide, isolating or producing a substantially purified calcium- independent srtA mutant disclosed herein, administering sortagged cells, e.g., implanting or transplanting in vivo, or screening for a compound or agent that modulates activity of the calcium- independent srtA mutants or variants thereof .

In certain embodiments, a packaged product or kit includes a container, such as a sealed pouch or shipping container, or an article of manufacture, for example, to carry out a sortase-mediated ligation reaction utilizing a calcium-independent srtA mutant described herein, variant thereof or composition comprising the same, or preserving or storing the calcium- independent srtA mutants , such as a tissue culture dish, tube, flask, roller bottle or plate (e.g., a single multi-well plate or dish such as an 8 , 16, 32 , 64, 96, 384 and 1536 multi- well plate or dish) .

The term "packaging material" refers to a physical structure housing the product or components of the kit . The packaging material can maintain the components sterilel , and can be made of material commonly used for such purposes (e.g. , paper, corrugated fiber, glass , plastic , foil , ampules , etc . ) . A label or packaging insert can be included, listing contents or appropriate written instructions, for example, practicing a method of the disclosure.

A packaged product or kit can therefore include instructions for practicing any of the methods of the disclosure described herein. For example, calcium- independent srtA mutants described herein, variants thereof, or enzyme compositions comprising them can be included in a tissue culture dish, tube, flask, roller bottle or plate (e.g., a single multi-well plate or dish such as an 8, 16, 32, 64, 96, 384 and 1536 multi-well plate or dish) together with instructions, e.g., for sortagging, purification, preserving or screening.

Instructions may be on "printed matter," e.g., on paper or cardboard within the kit, on a label affixed to the package, kit or packaging material, or attached to a tissue culture dish, tube, flask, roller bottle, plate (e.g., a single multi-well plate or dish such as an 8, 16, 32, 64, 96, 384 and 1536 multi-well plate or dish) or vial containing a component of the kit. Instructions may comprise voice or video tape and additionally be included on a computer readable medium, such as a disk (floppy diskette or hard disk) , optical CD such as CD- or DVD- ROM/RAM, magnetic tape, electrical storage media such as RAM and ROM and hybrids of these such as magnetic/optical storage media.

Disclosed kits can optionally include additional components, such as buffering agent, a preservative, or a reagent. Each component of the kit can be enclosed within an individual container or in a mixture and all of the various containers can be within single or multiple packages .

Further, some aspects of this disclosure provide that nucleophiles can be used in a sortase reaction that comprise reactive chemical moieties, for example, moieties, or "handles", suitable for a click chemistry- reaction, as is described in detail in Published PCT International Application WO 2013/036630, the entirety of which is incorporated herein by reference.

* * *

One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The details of the description and the examples herein are representative of certain embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The articles "a" and "an" as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include "or" between one or more members of a group are

considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. It is contemplated that all embodiments described herein are applicable to all different aspects of the invention where appropriate. It is also contemplated that any of the embodiments or aspects can be freely combined with one or more other such embodiments or aspects whenever appropriate. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element (s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as

comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist , or consist essentially of , such elements , features, etc. For purposes of simplicity those

embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims , regardless of whether the specific exclusion is recited in the

specification. For example, any one or more nucleic acids , polypeptides , cells , species or types of organism, disorders, subjects, or combinations thereof, can be excluded .

Where the claims or description relate to a

composition of matter, e.g., a nucleic acid, polypeptide, cell, or non-human transgenic animal, it is to be understood that methods of making or using the

composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter for any of the purposes disclosed herein are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where the claims or description relate to a method, e.g., it is to be understood that methods of making compositions useful for performing the method, and products produced according to the method, are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise.

Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the

invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Numerical values, as used herein, include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by "about" or "approximately" , the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by "about" or "approximately" , the invention includes an embodiment in which the value is prefaced by "about" or "approximately" . "Approximately" or "about" generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value) . It should be

understood that , unless clearly indicated to the

contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes

embodiments in which the order is so limited. It should also be understood that unless otherwise indicated or evident from the context, any product or composition described herein may be considered "isolated" . Examples

Example 1: A substantially pure, stable, high yield, calcium-independent srtA monomeric mutant

The present inventors surprisingly and unexpectedly found that a calcium- independent srtA mutant (SEQ ID NO: 9) purifies predominantly in monomeric form compared to a calcium-dependent srtA mutant (SEQ ID NO: 7) that purifies both in dimeric and monomeric forms.

The sortase purification process involves direct loading of clarified E.coli lysate onto a nickel NTA column. His-tagged sortase binds to the nickel NTA column (via metal chelation) and is eluted using an imidazole buffer. Eluted material from the nickel NTA column is loaded onto a size exclusion chromatography column (SEC) (e.g., a HILOAD^® 16/600 SUPERDEX^® 75 pg) and resulting separations (chromatogram) are show in FIGS. 7A, 7B and 7C.

The column resolves/separates residual high

molecular weight impurities from the sortase dimer and monomer. As is shown in FIGS. 7A, 7B and 7C,

unexpectedly, the calcium- independent srtA mutant of SEQ ID NO: 9 exists predominantly in monomeric form (FIG. 7A) , in contrast, as noted above, the calcium-dependent sortase obtained from SEQ ID NO: 7 exists in both dimeric and monomeric form (FIG. 7B) , as does the calcium- dependent wild-type sortase (FIG. 7C) . Those skilled in the art will appreciate the unexpected advantages which this confers on the calcium- independent srtA mutant of SEQ ID NO: 9, including a higher recovery yield of 100% monomer, and simpler manufacturing process to isolate monomer as there is far less dimer to separate out, amongst others. Without wishing to be bound by theory, it is believed that the lower amount of dimer form observed with the calcium- independent srtA mutant of SEQ ID NO: 9 is due to lower propensity to dimerize based on the outer surface location of the substituted amino acids between the calcium-dependent srtA mutant of SEQ ID NO: 7 and the calcium- independent srtA mutant of SEQ ID NO: 9 (see, e.g., FIG. 5B for an alignment of SEQ ID NO: 7 and SEQ ID NO: 9). The lower propensity of the calcium-independent srtA mutant of SEQ ID NO: 9 to dimerize is likely to translate into a more "stable" enzyme as it should also have a lower tendency to dimerize during storage, which may prolong shelf-life or permit storage under less costly conditions (e.g. refrigerated vs. frozen at - 80°C) .

FIG. 8 shows a gel demonstrating the high purity of monomeric sortase compared to dimeric sortase. For example, the gel illustrates the protein constituents present in the three main components from the calcium- independent srtA mutant of SEQ ID NO: 9 preparation separated on the SEC purification column. Collected fractions were pooled comprising the Void, Dimer and Monomeric peaks shown in FIG. 7B. The "Load" in FIG. 8 represents the starting material in the clarified E. coli lysate. The "Void" lane in FIG. 7B contains some sortase along with a large number of higher molecular weight proteins. The "Dimer" lane in FIG. 8 contains

predominantly sortase but also contains some higher molecular weight proteins. The "Monomer" lane in FIG. 8 runs as a single sortase band on the gel, indicating high purity of the monomeric form of the calcium- independent srtA mutant of SEQ ID NO: 9. FIG . 9 shows the SEC results demonstrating the stability of monomeric sortase. Briefly, the dimeric and monomeric pools collected from the SEC purification step described above were stored at 4 °C for 24 hours, and then analyzed by analytical SEC (SUPERDEX^® 75 10/300 GL) . As shown in FIG. 9, the monomeric sortase isolated during the initial purification, when analyzed one day later, still exists in monomeric form indicating that the monomeric form is stable and not in dynamic equilibrium with the dimer. Similarly, the dimeric sortase isolated during the initial purification, when analyzed one day later, still exists in dimeric form. One can note a small amount of monomer in the Dimer Pool as a

consequence of pooling a fraction that was not completely resolved (separated) in the SEC purification step.

Taken together, the work described herein

demonstrates that the calcium- independent srtA mutant of SEQ ID NO: 9 can be utilized to produce high yields of a substantially pure, stable, monomeric sortase enzyme that exhibits increased sortase A catalytic activity compared to wild-type srtA regardless of the availability of calcium .

Example 2 : Exemplary Method of Generating Calcium- Independent Sortase A Mutants

As noted above , given the nucleotide and amino acid sequences of the calcium- independent srtA mutants disclosed herein (e.g., SEQ ID NOs : 9-21) , a skilled artisan can readily appreciate a variety of suitable techniques for generating calcium- independent srtA mutants based on those sequences , e.g., site-directed mutagenesis , in vitro translation of synthetic mRNA, de novo synthesis , for example , utilizing solid phase peptide synthesis . What follows is a description of an exemplary method of generating a calcium- independent srtA mutant comprising SEQ ID NO: 9. The skilled artisan will appreciate that the method can readily be adapted to generate any calcium- independent srtA mutant. Other suitable methods will be apparent to the skilled artisan based on the teachings herein.

The calcium- independent srtA mutant of SEQ ID NO: 9 was generated by introducing two additional nucleotide point mutations into a calcium-dependent srtA mutant of SEQ ID NO: 7. These nucleotide point mutations comprised a G->A mutation at position 139, and a G-C mutation at position 148 of SEQ ID NO: 7 .

The mutations were introduced on primers used to amplify a pET29 mutant delta 59 SrtA construct (see, e.g., Liu et al . , "A general strategy for the evolution of bond-forming enzymes using yeast display," PNAS.

2011; 108 (28) : 11399-11404) . PCR amplification was then be performed in two stages. First, the construct was amplified with primers shown in Table 4 below such that the forward primer introduced a restriction site for cloning (e.g., a Ndel restriction site), and the reverse primer introduced the two point mutations described above .

Table 4 : Primers for first half of the construct

Next, the second half of the construct was amplified with the primers shown in Table 5 below such that the forward primer introduced the two point mutations described above, and the reverse primer introduced a restriction site for cloning (e.g., a Xhol restriction site) .

Table 5: Primers for second half of construct

The PCR reactions were performed using the pET29 pentamutant delta 59 SrtA plasmid DNA as a template, under the conditions set forth in Table 6 and cycling conditions set forth in Table 7 below.

Table 6: PCR Reaction Conditions for First Round of

PCR

0.5uL Template DNA (l-2ng total)

5uL lOx HiFi Polymerase buffer (Roche)

1.5uL lOuM forward primer

1.5uL lOuM reverse primer

luL HiFi Polymerase

1.2uL lOmM dNTPs

6uL 25mM MgC12

33uL dH20

Table 7: PCR Cycling Conditions for First Round of

PCR

95C for 5min

35 cycles of

95C for 30s

55C for 30s

72C for 1 min 72C for 7min

4C

The resulting PGR products from the first round of PGR were analyzed by gel electrophoresis, excised from the gel and purified. The products were then used as a template in a second round of PGR, and amplified using forward primer 1 and reverse primer 2 described above. The second round of PCR reactions was carried out under the conditions set forth in Table 8 below under the cycling conditions set forth in Table 9.

Table 8 : PCR Reaction Conditions for First Round of

PCR

5uL PCR product 1

luL PCR product 2

5uL lOx HiFi Buffer (Roche)

6uL 25mM MgCl2

1.2uL 10m dNTPs

luL HiFi Polymerase

30.8uL dH20

Table 9 : PCR Cycling Conditions for First Round of

PCR 95C for 5min

followed by 5 cycles of

95C 30s

55C 30s

72C 1 min

At this point, the following was added to the

reaction :

luL HiFi Polymerase (Roche)

1.5uL lOuM forward primer

1.5uL lOuM reverse primer

And cycled as follows:

95C for 5min

35 cycles of

95C for 30s

55C for 30s

72C for 1 min

72C for 7min

4C

The resulting product from the second round of PGR reactions was analyzed by gel electrophoresis, digested with Ndel and Xhol , and ligated into pET30b so that there is 6x his tag at the 3' end of the construct.

The construct was then verified by DNA sequencing and transformed into chemically competent BL21 (DE3) E.coli for protein expression. Protein was expressed and purified following published methods (Popp, Current Protocols in Protein Science, 2009) . Sequence Key

SEQ ID NO: 1 - Exemplary Wild Type S. aureus Sortase A amino acid sequence

SEQ ID NO: 2 - Nucleotide sequence encoding Wild Type S. aureus Sortase A

SEQ ID NO: 3 - Exemplary Wild Type S. aureus Sortase A amino acid sequence

SEQ ID NO: 4 - Nucleotide sequence encoding Wild Type S. aureus Sortase A

SEQ ID NO: 5 - WT sortase delta 25 amino acid sequence

SEQ ID NO: 6 - Nucleotide sequence encoding WT sortase delta 25

SEQ ID NO: 7 - srtA P94R/D160N/D165A/K190E/K196T amino acid sequence

SEQ ID NO: 8 - Nucleotide sequence encoding srtA

P94R/D160N/D165A/K190E/K196T

SEQ ID NO: 9 - Calcium- independent srtA

P94R/D160N/D165A/K190E/K196T

SEQ ID NO: 10 - Nucleotide sequence encoding calcium- independent srtA P94R/D160N/D165A/K190E/K196T

SEQ ID NO: 11 - Calcium- independent srtA

P94S/D160N/D165A/K196T amino acid sequence

SEQ ID NO: 12 - Calcium- independent srtA

D160N/K190E/K196T amino acid sequence

SEQ ID NO: 13 - Calcium- independent srtA P94S/D160N/K196T amino acid sequence

SEQ ID NO: 14- Calcium- independent srtA P94S/D160N/D165A amino acid sequence

SEQ ID NO: 15 - Calcium- independent srtA P94S/D165A amino acid sequence

SEQ ID NO: 16 - Calcium- independent srtA P94S amino acid sequence

SEQ ID NO: 17 - Calcium- independent srtA P94R amino acid sequence

SEQ ID NO: 18 - Calcium- independent srtA D160N amino acid sequence

SEQ ID NO: 19 - Calcium- independent srtA D165A amino acid sequence SEQ ID NO 20 - Calcium- independent srtA K190E amino acid sequence

SEQ ID NO 21 - Calcium- independent srtA K196T amino acid sequence

SEQ ID NO 22 - Forward Primer 1

SEQ ID NO 23 - Reverse Primer 1

SEQ ID NO 24 - Forward Primer 2

SEQ ID NO 25 - Reverse Primer 2

Claims

CLAIMS What is claimed is:

1. A sortase A mutant comprising at least three amino acid substitutions relative to a wild-type sortase A, wherein the amino acid substitutions comprise a) a K residue at position 105; b) a Q or A residue at position 108; and c) at least one amino acid substitution selected from the group consisting of i) a R residue at position 94; ii) a S residue at position 94; iii) a N residue at position 160; iv) a A residue at position 165; v) a E residue at position 190; and vi) a T residue at position 196.

2. The mutant of claim 1, wherein the wild- type sortase A comprises a S. aureus sortase A

3. The mutant of claim 1, wherein the wild- type sortase A comprises a protein sequence of SEQ ID NO: 1.

4. The mutant of claim 1 , wherein the wild- type sortase A comprises a protein sequence of SEQ ID NO: 3.

5. The mutant of any one of claims 1 to 4, comprising a deletion of amino acids 2-25.

6. The mutant of any one of claims 1 to 5, comprising a deletion of amino acids 2-59.

7. The mutant of any one of claims 1 to 6, comprising at least two amino acid substitutions selected from the group consisting of i) -vi) .

8. The mutant of any one of claims 1 to 7 , comprising at least three amino acid substitutions selected from the group consisting of i) -vi) .

9. The mutant of any one of claims 1 to 8 , comprising at least four amino acid substitutions selected from the group consisting of i) -vi) .

10. The mutant of any one of claims 1 to 9, comprising at least five amino acid substitutions selected from the group consisting of i) -vi) .

11. The mutant of any one of claims 1 to 10, wherein the mutant comprises at least 60% sequence identity to amino acid residues 60-206 of the wild-type sortase A.

12. The mutant of any one of claims 1 to 11, wherein the mutant comprises at least 80% sequence identity to amino acid residues 60-206 of the wild-type sortase A.

13. The mutant of any one of claims 1 to 12, wherein the mutant comprises at least 90% sequence identity to amino acid residues 60-206 of the wild-type sortase A.

14. The mutant of any one of claims 1 to 13, further comprising one or more C-terminal or N-terminal tags.

15. The mutant of claim 14, wherein the one or more C- terminal or N-terminal tags comprises a His6 tag.

16. The mutant of any one of claims 1 to 15, wherein the mutant exhibits sortase A catalytic activity in the absence of calcium.

17. The mutant of any one of claims 1 to 16, wherein the mutant exhibits sortase A catalytic activity in the absence of exogenous calcium.

18. The mutant of any one of claims 1 to 17, wherein the mutant exhibits sortase A catalytic activity in the presence of calcium-binding proteins.

19. The mutant of any one of claims 1 to 18, wherein the mutant exhibits sortase A catalytic activity in the presence of calcium concentrations up to 10 mM .

20. A polynucleotide encoding the mutant of any one of claims 1 to 19.

21. A nucleic acid construct comprising the

polynucleotide of claim 18.

22. A host cell transformed with the nucleic acid construct of claim 19.

23. A method of preparing a mutant sortase A comprising:

(a) culturing the host cell of claim 22 in a suitable culture medium under suitable conditions to produce the mutant sortase A; and optionally

(b) purifying the mutant sortase A to provide a mutant sortase A.

24. An enzyme composition comprising at least one sortase A mutant of any one of claims 1 to 19.

25. A method comprising performing a sortase-mediated transpeptidation reaction catalyzed by the enzyme composition of claim 24.

26. Use of an enzyme composition of claim 24 for the sortagging of a target protein.

27. A method for sortagging a target protein,

comprising :

(a) providing a target protein comprising a sortase recognition motif;

(b) providing a moiety conjugated to a terminal oligoglycine sequence or a terminal alkylamine ; and

(c) contacting the target protein with the moiety in the presence of the enzyme composition of claim 24 under conditions suitable for the sortase A mutant to

28. The method of claim 27, wherein the terminal oligoglycine sequence comprises 1-10 N-terminal glycine residues .

29. The method of claims 27 or 28, wherein the moiety comprises an amino acid, a peptide, a protein, a

polynucleotide, a carbohydrate, a tag, a metal atom, a chelating agent, a contrast agent, a catalyst, a polymer, a recognition element, a small molecule, a lipid, a label, an epitope, a small molecule, a therapeutic agent, a cross-linker, a toxin, a radioisotope, an antigen, or a click chemistry handle.

30. A kit for sortagging a target protein comprising the enzyme composition of claim 24.

31. A sortase A mutant comprising an amino acid sequence at least 80% identical to SEQ ID NO: 9, and

wherein the mutant comprises a) a K residue at position 47 of SEQ ID NO: 9; b) a Q or A residue at position 50 of SEQ ID NO: 9; and c) at least one amino acid residue of SEQ ID NO: 9 selected from the group consisting of i) a R residue at position 36 of SEQ ID NO: 9; ii) a N residue at position 102 of SEQ ID NO: 9; iii) a A residue at position 107 of SEQ ID NO: 9; iv) a E residue at position 132 of SEQ ID NO: 9; and v) a T residue at position 138 of SEQ ID NO: 9.

32. The mutant of claim 31, comprising an amino acid sequence of SEQ ID NO: 16.

33. The mutant of claim 31, comprising an amino acid sequence of SEQ ID NO: 17.

34. The mutant of claim 31, comprising an amino acid sequence of SEQ ID NO: 18.

35. The mutant of claim 31, comprising an amino acid sequence of SEQ ID NO: 19.

36. The mutant of claim 31, comprising an amino acid sequence of SEQ ID NO: 20.

37. The mutant of claim 31, comprising an amino acid sequence of SEQ ID NO: 21.

38. The mutant of claim 31, comprising at least two amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) .

39. The mutant of claim 38, comprising an amino acid sequence of SEQ ID NO: 15.

40. The mutant of claim 31, comprising at least three amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) .

41. The mutant of claim 40, comprising an amino acid sequence of SEQ ID NO: 12.

42. The mutant of claim 40, comprising an amino acid sequence of SEQ ID NO: 13.

43. The mutant of claim 40, comprising an amino acid sequence of SEQ ID NO: 14.

44. The mutant of claim 31, comprising at least four amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) .

45. The mutant of claim 44, comprising an amino acid sequence of SEQ ID NO: 11.

46. The mutant of claim 31, comprising an amino acid sequence at least 90% identical to SEQ ID NO: 9.

47. The mutant of claim 31, comprising an amino acid sequence at least 95% identical to SEQ ID NO : 9.

48. The mutant of claim 31, comprising an amino acid sequence at least 96% identical to SEQ ID NO: 9.

49. The mutant of claim 31, comprising an amino acid sequence at least 97% identical to SEQ ID NO: 9.

50. The mutant of claim 31, comprising an amino acid sequence at least 98% identical to SEQ ID NO : 9.

51. The mutant of claim 31, comprising an amino acid sequence at least 99% identical to SEQ ID NO: 9.

52. The mutant of claim 31, comprising an amino acid sequence of SEQ ID NO: 9.

53. An enzyme composition comprising at least one mutant sortase A of any one of claims 31 to 52.

54. A method comprising performing a sortase-mediated transpeptidation reaction catalyzed by the enzyme composition of claim 53.

55. Use of an enzyme composition of claim 53 for the sortagging of a target protein.

56. A method for sortagging a target protein,

comprising :

(a) providing a target protein comprising a sortase recognition motif ; (b) providing a moiety conjugated to at least one of an oligoglycine sequence or a terminal alkylamine ; and

(c) contacting the target protein with the moiety in the presence of the enzyme composition of claim 53 under conditions suitable for the sortase A mutant to

57. The method of claim 56, wherein the oligoglycine sequence comprises 1-10 N-terminal glycine residues.

58. The method of claims 55 or 56, wherein the moiety comprises an amino acid, a peptide, a protein, a

59. A kit for sortagging a target protein comprising the enzyme composition of claim 53.

60. A polynucleotide encoding a sortase A mutant comprising a nucleotide sequence at least 80% identical to SEQ ID NO. 10, wherein the nucleotide sequence encodes a) a K residue at position 47 of SEQ ID NO: 9; b) a Q or A residue at position 50 of SEQ ID NO: 9; and c) at least one amino acid residue of SEQ ID NO: 9 selected from the group consisting of i) a R residue at position 36; ii) a N residue at position 102; iii) a A residue at position 107; iv) a E residue at position 132; and v) a T residue at position 138.

61. The polynucleotide of claim 60, wherein the

polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) - v) .

62. The polynucleotide of claim 60, wherein the

polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) - v) .

63. The polynucleotide of claim 60, wherein the

64. The polynucleotide of claim 60, comprising a nucleotide sequence at least 85% identical to SEQ ID NO: 10.

65. The polynucleotide of claim 60, comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 10.

66. The polynucleotide of claim 60, comprising a nucleotide sequence at least 95% identical to SEQ ID NO: 10.

67. The polynucleotide of claim 60, comprising a nucleotide sequence at least 96% identical to SEQ ID NO:

10.

68. The polynucleotide of claim 60, comprising a nucleotide sequence at least 97% identical to SEQ ID NO: 10.

69. The polynucleotide of claim 60, comprising a nucleotide sequence at least 98% identical to SEQ ID NO: 10.

70. The polynucleotide of claim 60, comprising a nucleotide sequence at least 99% identical to SEQ ID NO: 10.

71. The polynucleotide of claim 60, comprising a nucleotide sequence of SEQ ID NO: 10.

72. A nucleic acid construct comprising the

polynucleotide of any one of claims 60 to 71.

73. The nucleic acid construct of claim 72, further comprising a nucleotide sequence that encodes one or more C-terminal or N-terminal tags.

74. The nucleic acid construct of claim 73, wherein one or more C-terminal or N-terminal tags comprises a His6 tag.

75. A host cell transformed with the nucleic acid construct of any one of claims 72 to 74.

76. A method of preparing a mutant sortase A comprising : (a) culturing the host cell of claim 75 in a suitable culture medium under suitable conditions to produce the mutant sortase A; and optionally (b) purifying the mutant sortase A to provide a mutant sortase A .