WO2008065673A1

WO2008065673A1 - A method for producing n-terminal methioine free polypeptides in microbial host cells

Info

Publication number: WO2008065673A1
Application number: PCT/IN2007/000012
Authority: WO
Inventors: Kedarnath Sastry; Akundi Venkata Sriram; Anuj Goel; Sandeep Vishwanath Kamath; Hardik Valera; Mayank Kumar Garg; Suma Sreenivas; Malur Dattatreya Swetha; Amrita Basu; Reena Nichinmetla Raghunandan
Original assignee: Biocon Limited
Priority date: 2006-11-30
Filing date: 2007-01-12
Publication date: 2008-06-05
Also published as: EP2097520A4; EP2097520A1; MX2009005764A

Abstract

A method of making N-terminal methionine free polypeptide in a recombinant microbial host cell and pharmaceutical compositions along with one or more pharmaceutical excipients for management of various therapeutic disorders. Specifically, the invention relates to the production of N-terminal methionine free Streptokinase or human growth hormone by co expression with a protease enzyme in E.coli.

Description

A METHOD FOR PRODUCING N-TERMINAL METHIOINE FREE

POLYPEPTIDES IN MICROBIAL HOST CELLS Field of lhe Invention

The present invention provides a method of obtaining a N-terminal methionine free polypeptides in E.coli by constructing a plasmid to express a Ubiquitin like protein SMT3 -polypeptide fusion protein in the E.coli cytoplasm, which is cleaved by the co- expressed ULPl protease. The SMT3 -polypeptide fusion protein of the instant invention is specifically a SMT3 -Streptokinase, a SMT3 -Streptokinase fusion protein. Background and prior art of the Invention Streptokinase is a thrombolytic drug approved for use in acute myocardial infarction and thromboembolism. Streptokinase acts on plasminogen to convert it into plasmin which in turn degrades fibrin into soluble products. Streptokinase C (SKC) is synthesized as a precursor protein of 440 amino acids from which a 26 amino acid signal sequence is cleaved off before secretion from hemolytic Streptococci. Both native (non-recombinant) and recombinant SKC (rSKC) are commercially available. In two recent studies it was reported that, of the sixteen SKC preparations available in the market, only three showed potency fulfilling the criteria established by European Pharmacopoeia (Longstaff C, Thelwell C, and Whitton C, J. Thromb. Haemost., 3: 1092-1093, 2005 and Hermentin P, Cuesta-Linker T, Weisse, Schmidt K-H, Knorst M, Scheid M and Thimme M, European Heart Journal, 26:933-940, 2005). Discrepancies were observed in potency of many marketed rSKC as measured by in vitro assays as compared to a reference standard. Among the three rSKC samples included in the study, two showed potency ranging from 20.8% to 37.2% of the label claim, while a third rSKC preparation from China showed abnormal reaction kinetics and hence potency could not be determined at all. The N-termini of rSKC was found to be variable. While one rSKC preparation from Cuba showed approximately 50% of molecules with correct N-termini beginning with isoleucine, while the other 50% still had intact methionine at N-termini. An rSKC preparation from China had 6 extra amino acids at the N-terminus, presumably from the signal sequence. The third rSKC from India gave two sequences, both of which had no relationship to SKC N-terminus. Native SKC has the following amino acid sequence at N-terminus: IAGPEWLLDRPSVNH.

Currently SKC potency evaluation is done by two in-vitro assays - one in the absence of fibrin using a chromogenic substrate and another using fibrin as a substrate for clot- lysis where the activity is declared in international reference units. The value obtained by these two assays is identical when native streptokinase is tested. If the manufacturer of the recombinant SKC choose only one method (either chromogenic or clot-lysis assay) to determine potency, it is possible that the potency values obtained could be either be overestimated or underestimated. This under or over estimation has dramatic consequences during treatment. If the dose administered is lower than recommended, then reperfusion rate could be lower. Where as a high dose of streptokinase administered could cause increase intracranial hemorrhage. The N-terminus of Streptokinase plays a key role in Plasminogen activation during fibrin clot lysis Mundada LV, Prorok M, DeFord ME, Figuera M, Castellino FJ, Fay WPJ Biol Chem. 2003 JuI 4;278(27):24421-7. We have observed variation in the activity is up to two fold between the two assays for recombinant strepokinases with or without the N-terminal methionine. Thus there is a need to make a good quality recombinant SKC which is methionine free at its N-terminus, herein after called Met⁰ SKC, whose potency as measured by both chromogenic and clot-lysis assay are identical and comparable to properties of native SKC. The method used to manufacture Met⁰ SKC should be also simple, efficient and commercially viable. When recombinant proteins are expressed in E.coli, it is necessary to have ATG codon, encoding Methionine for initiation of protein synthesis. When heterologus proteins are expressed in E.coli, the N-terminal methionine may be removed in vivo, by methionine amino peptidase encoded by the genome of E.coli. In some cases, either due to primary or secondary structure, or inaccessibility of the N-terminus, or simply due to over expression, the N-terminal methionine fails to be removed. One method to solve this problem is to employ a tag at the N-terminus of the recombinant protein with suitable cleavage site to construct a fusion protein. ■ Methods for producing a recombinant protein with an N- or C-terminal tag are well known in the art. For therapeutic applications, the tag is removed from the purified recombinant protein by enzymatic cleavage in vitro, to generate the authentic N-terminus bearing protein. Many tags are available to the practitioners of art. An example of such a strategy is adding Glutathione S-transferase (GST) tag ending in C-terminus with amino acids He- Glu/Asp-Gly-Arg, which is cleaved specifically by Factor Xa protease, resulting in a recombinant protein with authentic N-terminus. This method is cumbersome as Factor Xa protease has to be produced separately and purified before its use in vitro for fusion protein cleavage. Also, many of the proteases also cleave the fusion protein at various non-canonical sites reducing overall yield of recombinant protein. The table below summarizes the current state of art, wherein only enterokinase, Factor Xa protease and intein tags allow cleavage in a specific manner, without leaving behind extra amino acids at the N-terminus.

The reason for the ever increasing number of tags made available for various N- terminus cleavages is due to the fact that it is not possible to predict whether a particular tag will provide the desired result. Two common reasons for using a tag are to improve the expression of a recombinant protein or make the expressed recombinant protein soluble. Often, recombinant heterologus proteins expressed in E.coli cytoplasm become insoluble forming the so called "inclusion bodies" and the use of tag may make the protein soluble at least partially. In other cases the tag may make the purification process simpler. For example adding a His tag to the expressed recombinant protein, allows simple and rapid purification using metal affinity chromatography step. One could also combine two tags, for example, His tag (comprising of six or ten Histidine residues) could be fused to another tag (for example, SMT3) which could make the recombinant protein more soluble. The advantage of His tag is easy purification from cytoplasm, using metal affinity chromatography. A second reason for adding tags to N-terminus is to make the proteins stable and prevent its degradation by proteases in E.coli cytoplasm. Small peptides/proteins with low molecular weight (< 10 kD) are often degraded rapidly when expressed by themselves in E.coli. For example, small peptides like GLP-I, insulin, etc have been shown to be stable only when they are expressed along with a tag at N-terminus. Small Ubiquitin related Modifier protein (SUMO), is a protein which is covalently attached post translationally to N-terminus of various proteins. SMT3 from Saccharomyces cerevisiae is an example of Small Ubiquitin like protein. Ubiquitin and Ubiquitin like proteins play an important role in various cell functions (nuclear metabolism, cell proliferation, autophagy, etc.) by modulating protein structure and function. ULPl protease releases SMT3 tag from proteins to which it is attached. Interestingly prokaryote like E.coli has neither SMT3 nor ULPl protease gene in its genome. Hence use of SMT3 tag in prokaryotes is much more convenient. There are also no proteases described in E.coli which could cleave SMT3. Also, ULPl protease, due to its high specificity can not cleave any E.coli protein and is thus not toxic to E.coli. Hence use of SMT3 tag in E.coli is much easier as compared to eukaryote like yeasts, in which there is a protease which cleaves SMT3 tag in the middle, after which due to alteration in three dimensional structure of the SMT3 protein; the C-terminal half SMT3 attached to a protein of interest fails to be cleaved by ULPl protease, unless one adds back N-terminal half SUMO to the cleavage reaction. Ubiquitin-like-specific protease 1 (ULPl) is deubiquitinating enzyme which cleaves the C-termini of SMT3 and deconjugate SMT3 from the side chains of lysines (Li, SJ. and Hochstrasser, M. (1999) Nature 398, 246-251). Three-dimensional structure of a complex between SMT3 and ULPl show that certain surface features of SMT3 are recognized by ULPl that make the interaction specific (Mossessova, E. and Lima, CD. (2000) MoI. Cell. 5, 865-876). The catalytic domain (amino acids 403-621) of ULPl is sufficient for deconjugation of SMT3 from fusion proteins and has been show to be functional when expressed in E.coli (Mossessova, E. and Lima, CD. (2000) MoI. Cell. 5, 865-876).

Objects of the present invention:

The principal object of the present invention is to produce N-terminal methionine free polypeptides in microbial host cells. Another object of the present invention is to produce N-terminal methionine free polypeptides in bacterial and fungal host cells.

Yet another object of the present invention is to produce N-terminal methionine free streptokinase in E.coli host cells.

Still another object of the present invention is to develop a method for the making of N-terminal methionine free polypeptides in microbial host cells.

Still another object of the present invention is to develop a method for the making of

N-terminal methionine free streptokinase in E.coli host cells.

Still another object of the present invention is to arrive at pure form of recombinant N- terminal methionine free streptokinase (Met⁰ SKC). Still another object of the present invention is to prepare a pharmaceutical composition comprising streptokinase (Met⁰ SKC) along with one or more pharmaceutically acceptable excipients.

Still another object of the present invention is to prepare a pharmaceutical composition comprising Met⁰ hGH along with one or more pharmaceutically acceptable excipients.

Statement of Invention:

A method of making N-terminal methionine free polypeptide in a recombinant microbial host cell comprising: (i) preparing an expression construct containing a DNA encoding a polypeptide of interest; (ii) preparing an expression construct containing a DNA encoding a protease enzyme (iii) cloning both the expression constructs of (i) and

(ii) into the host cell to obtain transformant; (iv) fermentatng the transformant and inducing co-expression; and (v) obtaining the N-terminal methionine free polypeptide; a method of making N-terminal methionine free streptokinase in E.coli host comprising: (i) preparing an expression construct containing a DNA encoding a SUMO- streptokinase flision protein; (ii) preparing an expression construct containing a DNA encoding a UIp 1 protease; (iii) cloning both the expression construct of (i) and (ii) into an E.coli host; (iv) fermentation of the transformants and inducing co- expression; and (v) obtaining the N-terminal methionine free streptokinase by lysing the cells and purifying the protein; a substantially pure recombinant N-terminal methionine free streptokinase (Met⁰ SKC); a pharmaceutical composition comprising streptokinase (Met⁰ SKC) which is substantially free of Met-SKC along with one or more pharmaceutically acceptable excipients; and a pharmaceutical composition comprising Met⁰ hGH which is substantially free of N-terminal methionine-hGH along with one or more pharmaceutically acceptable excipients.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig.l. Map of the plasmid with SKC coding sequence driven by a T7 promoter sequence

Fig.2. SDS-PAGE analysis of total cell lysate from induced and uninduced cultures of

E.coli BL21 (DE3) transformed with pET SKC plasmid.

Fig.3. Map of the plasmid with mutated version of E.coli MAP coding sequence driven by native promoter. Fig.4. SDS-PAGE analysis of total cell lysate from induced and uninduced cultures of

E.coli BL21 (DE3) transformed with pET ULPl plasmid.

Fig.5. Schematic diagram showing method of construction of plasmid for co-expression of Saccharomyces cerevisiae ULPl in E.coli

Fig.6. Schematic representation of SMT3 and SKC gene fusion Fig.7 is a map of the plasmid with SMT3-SKC fusion coding sequence driven by a T7 promoter sequence

Fig.8. SDS-PAGE analysis of total cell lysates from induced and uninduced cultures of

E.coli Bill (DE3) co-transformed with pET SKC and pTrcULPl/pACYC plasmids.

Fig:9 Illustration explaining the soluble chromogen assay and clot lysis assay.

Description of Certain Preferred Embodiments of the Invention

The present invention is in relation to a method of making N-terminal methionine free polypeptide in a recombinant microbial host cell comprising: (i) preparing an expression construct containing a DNA encoding a polypeptide of interest; (ii) preparing an expression construct containing a DNA encoding a protease enzyme (iii)cloning both the expression constructs of (i) and (ii) into the host cell to obtain transformant;

(iv)fermentatng the transformant and inducing co-expression; and (v) obtaining the N-terminal methionine free polypeptide.

Another embodiment of the present invention wherein the microbial host cell is selected from a group comprising bacteria and fungi.

Yet another embodiment of the present invention wherein N-terminal methionine free polypeptide is btained either by secrection or by lysing the cells and purifying the protein.

Still another embodiment of the present invention wherein the polypeptide of interest is a hormone, an enzyme or a therapeutic protein.

Still another embodiment of the present invention wherein the hormone is a growth hormone, luteinising hormone, or a natriuretic peptide

Still another embodiment of the present invention wherein the therapeutic enzyme is streptokinase Still another embodiment of the present invention wherein the growth hormone is hGH The present invention is in relation to a method of making N-terminal methionine free streptokinase in E.coli host comprising:

(i) preparing an expression construct containing a DNA encoding a SUMO- streptokinase fusion protein; (ii) preparing an expression construct containing a DNA encoding a Ulpl protease;

(iii)cloning both the expression construct of (i) and (ii) into an E.coli host; (iv) fermentation of the transformants and inducing co-expression; and (v) obtaining the N-terminal methionine free streptokinase by lysing the cells and purifying the protein. Another embodiment of the present invention wherein the E.coli host is a BL21 or BL26 strain

Yet another embodiment of the present invention wherein the expression construct is a pET vector containing a DNA encoding SMT3-Streptokinase fusion protein. Still yet another embodiment of the present information, wherein the expression construct^ is a pACYC184 vector containing a DNA encoding UIp 1 protease operationally linked to a promoter.

Still yet another embodiment of the present information wherein the DNA encoding UIp 1 protease operationally linked to a promoter is integrated into E.coli genome.

Still yet another embodiment of the present information wherein N-terminal methionine free streptokinase is Met⁰ SKC of Seq. ID no. 17, with potency identical to native SKC. The present invention is in relation to a substantially pure recombinant N-terminal methionine free streptokinase (Met⁰ SKC) The present invention is in relation to a pharmaceutical composition comprising streptokinase (Met⁰ SKC) which is substantially free of Met-SKC along with one or more pharmaceutically acceptable excipient

The present invention is in relation to a pharmaceutical composition comprising Met⁰ hGH which is substantially free of N-terminal methionine-hGH along with one or more pharmaceutically acceptable excipients.

Another embodiment of the present invention, wherein the pharmaceutical excipients are selected from a group comprising granulating agents, binding agents, lubricating agents, disintegrating agents, sweetening agents, coloring agents, flavoring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents and spheronization agents.

Recombinant SKC when expressed in E.coli without a tag at the N-terminus results in a mixture of molecules with and without Methionine. The physicochemical properties of the rSKC molecules with and without Methionine are very similar and are hence difficult to separate by simple techniques. In the current invention, we have genetically engineered a SMT3 tag at the N-terminus of SKC to generate fusion protein, which is cleaved by the co-expressed ULPl protease resulting in Met⁰ SKC with correct N- terminus, matching that of native streptokinase. A vector has been developed in which PCR amplified SMT3 coding sequence has been fused in frame with PCR amplified coding sequence of mature SKC (Met⁰ SKC) and cloned into a vector in which the fusion protein sequence is operationally linked to phage T7 promoter. This recombinant vector is cloned to express the recombinant SMT3-Met° SKC fusion protein in a genetically engineered E.coli host like BL21 (DE3) or BL26 (DE3) strains, upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). The recombinant SMT3 -SKC fusion protein is expressed into the E.coli cytoplasm and is cleaved by the by co-expressed ULPl in vivo. The ULPl protease very specifically recognizes the three dimensional features of SMT3 protein, in addition to Gly-Gly sequence, in the C- terminus and cleaves the SMT3 to release Met⁰ SKC. This simplifies the method of cleavage as opposed to in vitro cleavage which requires a second fermentation to produce ULPl protease. The ULPl protease is expressed in the same cell by a compatible, low copy number recombinant plasmid vector into which PCR amplified coding sequence of ULPl protease is cloned operationally linked to tac promoter which is also inducible by IPTG. The instant invention allows a simple method of producing Met⁰ SKC in large quantities using recombinant techniques for use in pharmaceutical compositions. The alternate methods described in literature involve over expression of E.coli gene coding for methionine amino peptidase (MAP). MAP over expression does not always help in removal of methionine from the N-terminus for various reasons like the nature of penultimate amino acid, secondary structure of the protein near the N-terminus, etc. It has been previously reported that if the amino acid next to initiator methionine is Ala, GIy, Pro and Ser, it is easily cleaved by MAP. If the penultimate amino acid is He, VaI, Cys or Thr, then there is variably in cleavage and removal of methionine. Finally, if the penultimate amino acid is Lys, Arg, Leu, Asp, Asn, Phe, Met, Trp, Tyr, GIu, GIn or His, the initiator methionine is not removed. (Ben-Bassat A, Bioprocess Technology 12:147-159, 1991). In the case of SKC, penultimate amino acid is Isoleucine and hence we also found variability in removal of methionine by MAP

A recent report (Liao YD, Jeng JC, Wang CF, Wang SC, Chang ST., Protein Science. 13:1802-1810, 2004, US Patent 7109015) suggested that site-directed mutagenesis of three residues in E.coli methionine amino peptidase substrate binding pocket (Y168G, M206T, Q233G) allowed the removal methionine from proteins even if the penultimate residues were Met, His, Asp, Asn, GIu, GIn, Leu, He, Tyr, and Trp. However, in the case of SKC, neither over expression of the native MAP nor over expression of the mutated E.coli MAP (with Y168G, M206T & Q233G amino acid substitutions) failed to remove methionine from N-terminus of Met SKC completely, even though penultimate residue is He. SKC there fore appears to be unique protein, removal of methionine from which appears to be a challenge. Because of these constraints, we sought an alternate method in which close to 100% of the recombinant SKC molecules produce&would be methionine free.

SMT3 coding sequence was PCR amplified from genomic DNA of Saccharomyces cerevisiae. SKC mature form coding nucleic acid fragment was amplified from the genomic DNA of Streptococcus equisimilis. By employing overlapping primers, the C- terminal end of SMT3 (ending in amino acids Gly-Gly) was fused in frame to N- terminal of Met⁰ SKC (beginning with amino acids Ile-Ala). This fused PCR product was cloned into pET vector using which recombinant proteins could be made by induction with IPTG after transformation into appropriate commercially available genetically engineered E.coli host like BL21 (DE3), into whose genome lacUV5 promoter driven T7 phage polymerase coding sequence has been integrated (Novagen, Madison, USA). PCR technique was used to amplify ULPl protease gene from Saccharomyces cerevisiae genome and cloned into a low copy number vector (p AC YC) vector. The expression of ULPl was driven by tac promoter, which is also induced by IPTG. An alternative method of co-expression of ULPl protease by integration of the ULPl gene into E.coli genome is also possible. Preferably the ULPl gene could be targeted to msbB gene coding sequence, the disruption of which could cause a reduction in the levels of endotoxin from the E.coli host, helping in improving the quality of therapeutic protein by reduction of endotoxin impurity. The technology of the instant Application is further elaborated with the help of following examples. However, the examples should not be construed to limit the scope of the invention.

Example 1 Cloning and Expression ofrSKC in E.coli.

Preparation of replicative plasmid for intracellular production of Streptokinase in E.coli

Amplification of SKC gene: The source of DNA for mature Streptokinase encoding gene was the genomic DNA isolated from Streptococcus sp. strain BICC #7869 (MTCC 389). Genomic DNA was isolated using a commercially available (Qiagen) kit following manufacturer's instructions. The following primers were used for the amplification of the SKC gene. SKCFPl: 5' CATATGATTGCTGGACCTGAGTGGCTGCTA3' (Seq. ID#1) SKC RPl: 5' CGG GAT CCT TAT TTG TCG TTA GGG TTT ATC AGG 3' (Seq. ID#2).

Proof reading Pwo polymerase (Roche) was used for PCR and the SKC gene was amplified. The amplified product was electrophoresed on 1% agarose gel and was purified using the gel extraction kit (Qiagen). The gel purified product was modified to have dATP overhangs and this product was ligated to pTZ57R/T vector (MBI Fermentas).The ligation reaction was carried out at 16⁰C overnight. The ligation mix was transformed to chemically competent E.coli DH5α cells and plated on LB Ampicillin 100 μg/ml plates. The transformants were screened by isolating plasmid from 1 ml of culture by mini lysis method and was digested with restriction enzymes BamHI and Xbal (NEB) to release the insert. The digestion was carried out at 37⁰C for 2 hours. One positive clone was selected and inoculated to LB medium containing Ampicillin 100 μg/ml and plasmid was isolated by Qiagen mini prep kit and the plasmid was sequenced to confirm the presence of the gene and any possible mutations. The sequence confirmed plasmid was then used for further sub-cloning. Sub cloning SKC coding DNA fragment to pET vector: The plasmid which has the SKC gene in pTZ57R/T vector was digested with the restriction enzymes Ndel and BamHI and electrophoresed on 1% agarose gel. The insert released was excised and gel eluted and ligated to pET vector which was also digested with the same enzymes. The ligation was done using the enzyme T4 DNA ligase (NEB) and ligation was carried out at 16 ⁰C overnight. The ligation mix is used to transform competent E.coli DH5 alpha cells and the transformants were selected on LB plates with 100 μg/ml Ampicillin. The transformants are screened for the presence of the SKC gene. The correct clone (Fig. 1) was mapped and confirmed and this plasmid is used for transforming competent E.coli BL26 (DE3) host.

Expression in E.coli BL26 (DE3) host: The verified plasmid clone was transformed to E.coli BL26 (DE3) host and the transformants were selected on Ampicillin (100 μg/ml) plates. A few transformants were grown in LB Ampicillin (lOOμg/ml) liquid media and when the OD at 600nm is -0.6 to 0.8 the cells are induced with IPTG (1 mM). The induction is carried out overnight at 30 ⁰C and the cells were pelleted and resuspended in buffer and sonicated to lyse the cells. The samples were loaded on SDS-PAGE. The results are shown in Fig. 2. A good level of induction was observed. Recombinant SKC was purified and sequencing of the product showed that only 40% of the rSKC molecules were Met⁰ SKC.

Example 2. Over expression of E. coli MAP in E. coli expressing SKC

The main goal of this experiment was to determine if over expression of MAP in the same cell as that expressing rSKC would help in efficient removal of N-terminal methionine.

E.coli MAP gene was amplified from BL21 (DE3) host genomic DNA using the following primers (Seq. ID# 3 to Seq. ID# 10) to generate a mutated version which has been reported to remove N-terminal methionine more efficiently from proteins with He as the penultimate amino acid (Liao YD, Jeng JC, Wang CF, Wang SC, Chang ST., Protein Science. 13:1802-1810, 2004, US Patent 7109015). The mutations introduced were 168Y, 206M, 233Q.

Y168GFP 5'TTCGTGAAGGCTGCGGACACGGTATTGGTCGCGGS' Seq. ID# 3

Y168GRP 5'GTCCGCAGCCTTCACGAACGACGGAGAAGCCTTCS' Seq. ID# 4

M206TFP 5'CGAGCCAACCGTCAACGCGGGTAAAAAAGAGATCS' Seq. ID# 5

M206TRP 5'CGTTGACGGTTGGCTCGATGGTGAACGTCATCCCS' Seq. ID# 6

Q233GFP 5'GTCTGCAGGCTATGAGCATACTATTGTGGTGACTG3¹ Seq. ID# 7

Q233GRP 5'GCTCATAGCCTGCAGACAAGCTGCGATCTTTGGS' Seq. ID# 8

MetAPFP 5'GGATCCGACGTCGAATTTTCTATTAS' Seq. ID# 9

MetAPRP 5'TCGGGTATAGCTATGAAAGCAGCTGS' Seq. ID# 10

The primer positions have been represented in the following figure.

MetAPIP Y168GEP IM2C6TΪP Q233GFP

|Y168GKP |M20CTRP |Q233GRP |MetAPEP

The source of DNA for Methionine amino peptidase (MAP) was E.coli genomic DNA from BL21 (DE3) strain which was isolated by routine protocols (Molecular Cloning:

A Laboratory Manual By Joseph Sambrook, David W Russell, 2001, CSHL Press). Four separate PCR reactions were performed using E.coli genomic DNA as template and the proof-reading polymerase, Pwo polymerase with various primer pairs as outlined in the following table:

The four PCR reactions were run on 1.2% agarose gel for 1 hr at 100 mV. Individually the amplified fragments were excised from the gel and purified using Qiagen gel extraction kit. To get full length MAP gene (1048kb), all the four fragments were used as template using MetAPFP & MetAPRP primers. Proof reading Pwo polymerase (Roche) was used for PCR and the full length MAP gene was amplified. The amplified product was electrophoresed on 1% agarose gel and was purified using the gel extraction kit (Qiagen). The gel purified product was modified to have dATP overhangs and this product was ligated to pTZ57R/T vector (MBI Fermentas).The ligation reaction was carried out at 16⁰C overnight. The ligation mix was transformed to chemically competent E. coli DH5α cells, plated on LB Ampicillin (lOOμg/ml) plates incubated at 37 ⁰C, overnight. The transformants were screened by isolating plasmid from 1 ml of culture by mini lysis method and was digested with restriction enzymes BamHI and Xbal (NEB) to release the insert. The digestion was carried out at 37⁰C for 2 hours. One positive clone was selected and inoculated to LB broth containing Ampicillin (100 μg/ml), incubated at 37 ⁰C shaker incubator, overnight. Plasmid was isolated by Qiagen mini prep kit and the plasmid was sequenced to confirm the presence of the gene and mutations made at three positions.. The sequence confirmed plasmid was then used for further sub-cloning.

Sub-cloning MAP (Seq. ID #18) gene to pACYC vector; The plasmid with the MAP gene in pTZ57R/T vector was digested with the restriction enzymes Sail followed by BamHI and was electrophoresed on 1% agarose gel. The insert released was excised from the gel, eluted and ligated to pACYC vector which was also digested with the same enzymes sequentially. The ligation was done using the enzyme T4 DNA ligase (NEB) and ligation wais carried out at 16 ⁰C overnight. The ligation mix was used to transform competent E.coli DH5D cells and the transformants were selected on LB plates with Chloramphenicol (15μg/ml). The transformants were screened for the presence of the MAP gene. The correct clone was mapped and confirmed (Fig. 3).

Expression in E.coli BL26 (DE3) host: The verified plasmid clone was co- transformed along with pET SKC vector to E.coli BL26 (DE3) host and the transformants were selected on Ampicillin (100 μg/ml) and Chloramphenicol (15 μg/m)l plates. A few transformants were grown in LB broth with Ampicillin (lOOμg/ml) and Chloramphenicol (15μg/ml). When the OD at 600 nm was -0.6 to 0.8 the cells were induced with IPTG (1 mM). The induction was carried out overnight at 30 ⁰C and the cells were pelleted and resuspended in buffer and sonicated to lyse the cells. The samples were loaded on SDS-PAGE.

After studying the expression of rSKC (Seq. ID # 17) and purification, the results indicated that there was no improvement in efficiency of removal of methionine from N-terminus (data not shown). Approximately 40%-60% of the rSKC molecules still had methionine at their N-terminus. Example 3:

Cloning and Expression of SMT3-SKC fusion in E.coli The genomic DNA of Saccharomyces cerevisiae strain (BICC# 7806) was isolated using standard techniques. This was used as the template and a PCR reaction was carried out using the primers SMT FPl (5'

CATATGGGTCATCACCATCATCATCACGGGTCGG ACTCAGAAGTC 3', Seq. ID# 11) and SMT RPl (5 'CTCAGGTCC AGC AATACCTC CAATCTGTTCGCGGTG 3', Seq. ID# 12), the SMT-3 gene was amplified. The reaction mix was electrophoresed on 1.2% agarose gel. The amplified product was checked for the size and was gel eluted and was used as the template for overlapping PCR. In a similar way using the genomic DNA of the strain BICC 7869 \nά the primers SMT-SKC FPl (5' GAACAGATTGAAGGTATTGCTGGACCTGAGTGGCTG 3', Seq. ID# 13) and SMT-SKC RPl (5'CGG GAT CCT TAT TTG TCG TTA GGG TTT ATC AGG 3', Seq. ID# 14) the SKC gene was amplified by PCR using proof reading polymerase. This fragment was also gel eluted and used as template for overlapping PCR. By mixing both the purified PCR products and using them as the template, PCR was performed to get the full length overlapped product. This is electrophoresed on 1% agarose gel and the product is gel eluted. This is then ligated to pTZ57R/T vector by creating dATP overhangs and the ligation was carried out at 16°C overnight. The ligation mix is then transformed to competent E.coli DH5α cells and the mix was plated on LB containing Ampicillin (100μg/ml) plates and the plates were incubated at 37°C incubator overnight. The transformants which appear were screened for the presence of the gene and this plasmid was purified by using Qiagen mini prep kit. This plasmid was sequenced to confirm the overlap and for mutations. The confirmed plasmid was used for further experiments. This plasmid was digested with the restriction enzymes Ndel and BamHI (NEB) and the reaction mix was incubated at 37°C for 2 hours. The reaction was terminated by heat inactivation of the enzymes and it was electrophoresed on 1% agarose gel. The insert released was gel eluted and was used for ligation with the vector. The vector is prepared by digesting pET vector with the enzymes Ndel and BamHI and processed in a similar way as done to the insert. Then a ligation reaction was set using T4 DNA ligase (NEB) and the reaction was carried out at 16°C overnight. The ligation mix was then transformed to competent E.coli DH5α cells and then selected on LB Kanamycin (25μg/ml) plates. The transformants were screened for the presence of the gene and the plasmid was isolated from the correct clone using Qiagen Mini prep kit. The plasmid was confirmed by restriction analysis and was then used for expression studies. Schematic representation of SMT3 AND SKC genefusion is illustrated in Fig: 6. The plasmid containing SMT3-SKC fused gene (Fig: 7) product in pET vector was used to transform competent E.coli BL21 (DE3) cells. The transformants were selected on LB containing Kanamycin (25μg/ml) plates. A few colonies were inoculated to 100 ml LB containing Kanamycin (25μg/ml) and were grown to OD 600 of ~0.7-0.8. A small sample was removed as control and the rest of the culture was induced with IPTG to a final concentration of 1 mM. The induction was performed overnight at 30⁰C with shaking at 120 rpm. The cells were then pelleted and resuspended in buffer and sonicated to lyse the cells and the samples were analyzed on 10% SDS-PAGE. Example 4: Cloning and expression of UIpI protease in E.coli

Using the primers Ulpl FP (Ndel) 5' CAT ATG CTT GTT CCT GAA TTA AAT GAA AAA GAC G 3' (Seq. ID# 15) and Ulpl RP (Xhol) 5' CTC GAG TTT TAA AGC GTC GGT TAA AAT C 3' (Seq. ID# 16), the ULPl (Seq. Id # 19) gene was amplified by PCR using Saccharomyces cerevisiae genomic DNA as the template (BICC# 7806) and proof reading Pwo polymerase (Roche) enzyme. The PCR product was gel purified and cloned to pTZ57R/T vector (MBI Fermentas) after creating dATP overhangs. The ligation was carried over night at 16 °C. The ligation mix was transformed to competent E.coli DH5α cells and selected on LB Ampicillin (lOOμg/ml) plates. The colonies were screened by PCR using M 13 forward and M 13 reverse primers and gene specific forward and reverse primers. One correct clone was selected and plasmid from this clone was isolated by using Qiagen mini prep method. This clone was sequenced to confirm the presence of the gene and any possible mutations Results of sequencing of the PCR product showed three mutations. All three mutations were silent mutations (Asp⁴¹³ GAC to GAT, Ser⁴⁷³ TCA to TCG, GIu⁵⁵⁴ GAG to GAA). The correct confirmed clone was digested with restriction enzymes Ndel and Xhol (NEB) and the insert released was gel purified and ligated to the pET vector which was also digested with Ndel and Xhol restriction enzymes. The ligation was done using the enzyme T4 DNA ligase (NEB) and the reaction was carried out at 16⁰C overnight. The ligation mix was then transfoπned to competent E.coli DH5α cells by heat shock method. The transformation mix was then plated on LB plates carrying Kanamycin (25μg/ml) and the plates were incubated at 37⁰C overnight. The Kanamycin resistant transformants were screened for the presence of the gene and the correct clone was inoculated to LB medium with Kanamycin (25μg/ml) and plasmid was isolated by using Qiagen mini prep kit.

After confirming the authenticity of the plasmid by restriction digestions, it was transformed to chemically competent E.coli BL21 (DE3) competent cells and was plated on LB Kanamycin (25 μg/ml) plates.The plates were incubated at 37 ⁰C overnight. The Kanamycin resistant transformants were checked for protein production. A few Kanamycin resistant transformants were inoculated to 100 ml of liquid LB broth containing Kanamycin (25 μg/ml) and this was grown at 37⁰C till the OD at 600 nm reached 0.8. At this stage the culture was induced and a small amount of sample was removed as contol. Induction was done with IPTG (1 mM) and the culture was further grown at 30 ⁰C overnight. After overnight IPTG induction, the OD at 600 nm was checked and the cells were pelleted by centrifugation at 6000 rpm for 6 minutes. The cells were then resuspended in a buffer (100 mM Tris buffer, pH 8.0 with 0.1M NaCl) and sonicated using Branson sonifier till the OD dropped to 1/5 -1/8 of the initial OD. The soluble fraction was loaded on to Ni-NTA agarose matrix.(Qiagen) which was equilibrated with the same buffer. The matrix was washed with the same buffer and the protein was eluted from the matrix using the same buffer supplemented with 250 mM Imidazole. The results are shown in Fig. 5. A good level of induction (and UIp 1 production) was observed and the His-tagged UIp 1 was purified on Ni-NTA column chromatography. Example 5:

Co-expression of SMT3-SKC fusion protein and ULPl protease in E.coli (Sea Id # 201 Sub cloning of Ulpl to pTrc99A

The Ulpl protease encoding DNA fragment which was cloned to pET vector was digested with the restriction enzymes Ndel and BsrBI (NEB) and the digestion is carried out by incubating the reaction mix at 37⁰C. The reaction mix was then heated to high temperature to inactivate the enzymes and electrophoresed in l%agarose gel. The fragment released is then gel eluted and is ligated to pTrc99A vector which was also digested with the enzymes Ndel and Smal and was treated in similar manner. The ligation reaction was carried out using T4 DNA ligase (NEB) and the reaction was carried out at 16 ⁰C overnight. The ligated sample was then used to transform chemically competent E.coli DH5α cells and plated on LB Ampicillin (100g/ml) plates. The transformants which were resistant to Ampicillin (100g/ml) were screened for the presence of the insert by PCR using the gene specific primers. One correct clone was identified and used for further experiments. Sub cloning Ulpl coding sequence to pACYC184 vector: The Ulpl gene which was cloned to pTrc99A vector was digested with enzymes PvuII and BamHI (NEB) and the digestion was carried out at 37 ⁰C for 2 hours. The reaction was stopped by heat inactivating the enzymes and the mix was electrophoresed on 1% agarose gel. The insert released was gel purified and used for ligation with the vector. The pACYC184 vector was prepared by digesting with enzymes EcoRV and BamHI.The restriction enzyme digested pACYC184 vector was purified in a similar manner (Fig: 8). The insert and the vector were ligated using T4 DNA ligase. The ligated sample was transformed to competent DH5α cells and plated on LB agar plates containing Chloramphenicol (lOμg/ml). The plates were incubated at 37 ⁰C overnight. The Chloramphenicol resistant transformants were screened and the correct clone was confirmed by restriction analysis. This plasmid was used for further experiments. This pACYC184 vector with insert of UIp 1 gene in was co-transformed along with the plasmid harboring SMT3-SKC gene to chemically competent E.coli BL21 (DE3) host and plated on LB plates with Chloramphenicol (10 g/ml) & Kanamycin (25 g/ml).The plates were incubated at 37°C overnight. The transformants which appeared were resistant to both the antibiotics indicating the presence of both the plasmids. A few transformants were inoculated to 100 ml of LB medium containing both the antibiotics and the flask was incubated on a shaker at 37 ⁰C. When the OD at 600 nm reached ~0.7-0.8, a small sample was removed as control and the rest of the culture was induced with IPTG to a final concentration of 1 mM. The culture was next incubated at 30 ⁰C with shaking at 120 rpm overnight. The OD at 600 nm of the culture at the end of induction was checked and the cells were pelleted by centrifugation. The cell pellet was resuspended in a buffer containing 20 mM Tris, pH 8.0 and 100 mM NaCl. After resuspension the OD at 600 nm was checked and the volume was adjusted to -20-25 OD and the cells were sonicated using a cell disruptor, keeping on ice so that the temperature does not increase. The samples were loaded on 10% SDS-PAGE gel (Fig. 4). The cleavage was very efficient when both the protease and SMT3-SKC (Seq Id #21) were co expressed in the same host. The SKC was purified from lysed E.coli cells by standard purification methods. N-terminus of all the purified rSKC molecules was confirmed to be free of methionine.

Met SKC and Met free SKC were purified and analyzed for biological activity in solution chromogenic assay and Fibrin clear clot assay (Illustrated in Fig:9). The third international reference standard for Streptokinase (NIBSC Batch # 00/464 derived from Native Streptokinase) was used to calculate the relative potency and activity. The ratio of the activity for the two streptokinase preparations by these two methods was calculated and is presented in the table below

Batch number Met SKC Met free SKC

Batch #1 2.0 1.06

Batch #2 2.5 1.01

Batch #3 1.7 0.91

Seq. ID#1 CAT ATG ATT GCT GGA CCT GAG TGG CTG CTA Seq. ID#2

CGG GAT CCT TAT TTG TCG TTA GGG TTT ATC AGG

Seq. ID#3

TTC GTG AAG GCT GCG GAC ACG GTA TTG GTC GCG G

Seq. ID#4

GTCCGCAGCCTTCACGAACGACGGAGAAGCCTTC

Seq. ID#5

CGAGCCAACCGTCAACGCGGGTAAAAAAGAGATC Seq. ID#6

CGTTGACGGTTGGCTCGATGGTGAACGTCATCCC

Seq. ID#7

GTCTGCAGGCTATGAGCATACTATTGTGGTGACTG

Seq. ID#8 GCTCATAGCCTGCAGACAAGCTGCGATCTTTGG

Seq. ID#9

GGATCCGACGTCGAATTTTCTATTA

Seq. ID#10

TCGGGTATAGCTATGAAAGCAGCTG Seq. ID#l l

CATATGGGTCATCACCATCATCATCACGGGTCGGACTCAGAAGTC

Seq. ID#12

CTCAGGTCCAGCAATACCTCCAATCTGTTCGCGGTG

Seq. ID#13 GAACAGATTGAAGGTATTGCTGGACCTGAGTGGCTG

Seq. ID#14

CGG GAT CCT TAT TTG TCG TTA GGG TTT ATC AGG

Seq. ID#15

CAT ATG CTT GTT CCT GAA TTA AAT GAA AAA GAC G Seq. ID# 16

CTC GAG TTT TAA AGC GTC GGT TAA AAT C

Seq. ID# 17

Amino acid sequence of mature rSKC 1 IAGPEWLLDR PSVNNSLVVS VAGTVEGTNQ DISLKFFEID LTSRPAHGGK 51 TEQGLSPKSK PFATDSGAMS HKLEKADLLK AIQEQLIANV HSNDDYFEVI 101 DFASDATITD RNGKVYFADK DGSVTLPTQP VQEFLLSGHV RVRPYKEKPI 151 QNQAKSVDVE YTVQFTPLNP DDDFRPGLKD TKLLKTLAIG DTITSQELLA 201 QAQSILNKNH PGYTIYERDS SIVTHDNDIF RTILPMDQEF TYRVKNREQA

251 YRINKKSGLN EEINNTDLIS EKYYVLKKGE KPYDPFDRSH LKLFTIKYVD 301 VDTNELLKSE QLLTASERNL DFRDLYDPRD KAKLLYNNLD AFGIMDYTLT 351 GKVEDNHDDT NRIITVYMGK RPEGENASYH LAYDKDRYTE EEREVYSYLR 401 YTGTPIPDNP NDK

Seq. ID# 18

AMINO ACID SEQUENCE OF E.coli Methionine aminopeptidase (MAP) WITH

THREE MUTATIONS (Bold and underlined).

1 MAISIKTPED IEKMRVAGRL AAEVLEMIEP YVKPGVSTGE LDRICNDYIV

51 NEQHAVSACL GYHGYPKSVC ISINEVVCHG IPDDAKLLKD GDIVNIDVTV 101 IKDGFHGDTS KMFIVGKPTI MGERLCRITQ ESLYLALRMV KPGINLREIG 151 AAIQKFVEAE GFSVVREGCG HGIGRGFHEE PQVLHYDSRE TNVVLKPGMT 201 FTIEPTVNAG KKEIRTMKDG WTVKTKDRSL SAGYEHTIVV TDNGCEILTL 251 RKDDTIPAII SHDE

Seq. ID# 19

Nucleotide sequence of the ULPl PCR product amplified using Saccharomyces cerevisiae genome as template. The PCR product amplified nucleotides encoding amino acids 403-621 of ULPl protein. The silent nucleotide substitutions are shown in bold and underlined.

1 CATATGCTTG TTCCTGAATT AAATGAAAAA GACGATGATC AAGTACAAAA 51 AGCTTTGGCA TCTAGAGAAA ATACTCAGTT AATGAATAGA GATAATATAG 101 AGATAACAGT ACGTGATTTT AAGACCTTGG CACCACGAAG ATGGCTAAAT 151 GACACTATCA TTGAGTTTTT TATGAAATAC ATTGAAAAAT CTACCCCTAA

201 TACAGTGGCG TTTAATTCGT TTTTCTATAC CAATTTATCA GAAAGGGGTT 251 ATCAAGGCGT CCGGAGGTGG ATGAAGAGAA AGAAGACACA AATTGATAAA 301 CTTGATAAAA TCTTTACACC AATAAATTTG AACCAATCCC ACTGGGCGTT 351 GGGCATAATT GATTTAAAAA AGAAAACTAT AGGTTACGTA GATTCATTAT 401 CGAATGGTCC AAATGCTATG AGTTTCGCTA TACTGACTGA CTTGCAAAAA

451 TATGTTATGG AAGAAAGTAA GCATACAATA GGAGAAGACT TTGATTTGAT 501 TCATTTAGAT TGTCCGCAGC AACCAAATGG CTACGACTGT GGAATATATG 551 TTTGTATGAA TACTCTCTAT GGAAGTGCAG ATGCGCCATT GGATTTTGAT 601 TATAAAGATG CGATTAGGAT GAGAAGATTT ATTGCCCATT TGATTTTAAC 651 CGACGCTTTA AAACTCGAG

Seq. ID# 20 Amino acid sequence of the ULPl gene fragment expressed in E.colϊ.

1 ML VPELNEKD DDQ VQKALAS RENTQLMNRD NIEITVRDFK TLAPRRWLND 51 TIIEFFMKYI EKSTPNTVAF NSFFYTNLSE RGYQGVRRWM KRKKTQIDKL ioi DKiFTPiNLN QSHWALGIID LKKKTIGYVD SLSNGPNAMS FAILTDLQKY

151 VMEESKHTIG EDFDLIHLDC PQQPNGYDCG IYVCMNTLYG SADAPLDFDY 201 KDAIRMRRFI AHLILTDALK LEHHHHHHHH

Seq. ID# 21

Amino acid sequence of SMT3-SKC fusion protein. The sequence of SMT3 is shown in bold letters. 1 MGHHHHHHGS DSEVNQEAKP EVKPEVKPET HINLKVSDGS SEIFFKIKKT

51 TPLRRLMEAF AKRQGKEMDS LRFLYDGIRI QADQAPEDLD MEDNDIIEAH 101 REQIGGIAGP EWLLDRPSVN NSQLVVSVAG TVEGTNQDIS LKFFEIDLTS 151 RPAHGGKTEQ GLSPKSKPFA TDSGAMSHKL EKADLLKAIQ EQLIANVHSN 201 DDYFEVIDFA SDATITDRNG KVYFADKDGS VTLPTQPVQE FLLSGHVRVR 251 PYKEKPIQNQ AKSVDVEYTV QFTPLNPDDD FRPGLKDTKL LKTLAIGDTI

301 TSQELLAQAQ SILNKNHPGY TIYERDSSIV THDNDIFRTI LPMDQEFTYR 351 VKNREQAYRI NKKSGLNEEI NNTDLISEKY YVLKKGEKPY DPFDRSHLKL 401 FTIKYVDVDT NELLKSEQLL TASERNLDFR DLYDPRDKAK LLYNNLDAFG 451 IMDYTLTGKV EDNHDDTNRI ITVYMGKRPE GENASYHLAY DKDRYTEEER 501 EVYSYLRYTG TPIPDNPNDK

Claims

We Claim:

1. A method of making N-terminal methionine free polypeptide in a recombinant microbial host cell comprising:

(i) preparing an expression construct containing a DNA encoding a polypeptide of interest;

(ii) preparing an expression construct containing a DNA encoding a protease enzyme

(iii) cloning both the expression constructs of (i) and (ii) into the host cell to obtain transformant; (iv) fermentatng the transformant and inducing co-expression; and

(v) obtaining the N-terminal methionine free polypeptide.

2. The method as claimed in claim 1, wherein the microbial host cell is selected from a group comprising bacteria and fungi.

3. The method as claimed in claim 1, wherein N-terminal methionine free polypeptide is obtained either by secrection or by lysing the cells and purifying the protein.

4. The method in claim (i) of 1, wherein the polypeptide of interest is a hormone, an enzyme or a therapeutic protein.

5. The method in claim 2, wherein the hormone is a growth hormone, luteinising hormone, or a natriuretic peptide

6. The method in claim 4, wherein the therapeutic enzyme is streptokinase

7. The method in claim 4, wherein the growth hormone is hGH

8. A method of making N-terminal methionine free streptokinase in E.coli host comprising:

(i) preparing an expression construct containing a DNA encoding a SUMO- streptokinase fusion protein;

(ii) preparing an expression construct containing a DNA encoding a UIp 1 protease;

(iii) cloning both the expression construct of (i) and (ii) into an E.coli host;

(iv) fermentation of the transformants and inducing co-expression; and (v) obtaining the N-terminal methionine free streptokinase by lysing the cells and purifying the protein.

9. The method according to claim 8, wherein the E.coli host is a BL21 or BL26 strain.

10. The method according to claim (i) of 8, wherein the expression construct is a pET vector containing a DNA encoding SMT3 -Streptokinase fusion protein.

11. The method according to claim (ii) of 8, wherein the expression construct is a pACYC184 vector containing a DNA encoding UIp 1 protease operationally linked to a promoter.

12. The method according to claim (ii) of 8, wherein the DNA encoding Ulpl protease operationally linked to a promoter is integrated into E.coli genome.

13. The method according to claim (v) of 8, wherein N-terminal methionine free streptokinase is Met⁰ SKC of Seq. ID no. 17, with potency identical to native SKC.

14. A substantially pure recombinant N-terminal methionine free streptokinase

(Met⁰ SKC).

15. A pharmaceutical composition comprising streptokinase (Met⁰ SKC) which is substantially free of Met-SKC along with one or more pharmaceutically acceptable excipients.

16. A pharmaceutical composition comprising Met⁰ hGH which is substantially free of N-terminal methionine-hGH along with one or more pharmaceutically acceptable excipients.

17. The pharmaceutical compositions as claimed in claim 15 and 16, wherein the pharmaceutical excipients are selected from a group comprising granulating agents, binding agents, lubricating agents, disintegrating agents, sweetening agents, coloring agents, flavoring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents and spheronization agents.