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WO1998023737A1 - PRODUCTION DE FRAGMENTS D'ANTICORPS SYNTHETIQUES, A CHAINE UNIQUE, ETIQUETES ET $i(IN VIVO) - Google Patents

PRODUCTION DE FRAGMENTS D'ANTICORPS SYNTHETIQUES, A CHAINE UNIQUE, ETIQUETES ET $i(IN VIVO) Download PDF

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WO1998023737A1
WO1998023737A1 PCT/NL1997/000653 NL9700653W WO9823737A1 WO 1998023737 A1 WO1998023737 A1 WO 1998023737A1 NL 9700653 W NL9700653 W NL 9700653W WO 9823737 A1 WO9823737 A1 WO 9823737A1
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nucleic acid
scfv
acid molecule
protein
recombinant nucleic
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PCT/NL1997/000653
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WO1998023737A9 (fr
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Jasper Joris Van Thor
Klaas Jan Hellingwerf
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Universiteit Van Amsterdam
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Priority to AU54160/98A priority Critical patent/AU5416098A/en
Publication of WO1998023737A1 publication Critical patent/WO1998023737A1/fr
Publication of WO1998023737A9 publication Critical patent/WO1998023737A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to immunological reagents produced as fusion proteins that comprise single chain antibody fragments and reporter molecules or fragments thereof, which can for example be used in immunoassays .
  • Diagnostic as well as other applications of antibodies are numerous.
  • Both polyclonal as well as monoclonal antibodies (Mabs) have been used as immunological reagents to specifically bind to and detect antigens by a wide array of immunochemical techniques.
  • Such techniques such as enzyme- linked-immunosorbent-assays (ELISA), immunofluorescent assays (IFA), radio-immuno-assays (RIA), and others, are widely known in the field of immunological detection.
  • ELISA enzyme- linked-immunosorbent-assays
  • IFA immunofluorescent assays
  • RIA radio-immuno-assays
  • These techniques require the labeling or conjugation of the antibodies used with reporter molecules such as (fluorescent) dyes, enzymes, amino acid sequences, radio
  • Mabs are versatile immunological reagents for diagnostic purposes and are for instance used in a large array of detection kits. Mabs owe their usefulness to their highly specific and high-affinity binding to an antigen.
  • the V H and V L domain are the independently folded N- terminal variable domains of the H(eavy) and L(ight) chain of an antibody molecule and jointly they form the very specific antigen-binding site.
  • Mabs can be generated in a well known process by fusion of a selected lymphocyte (obtained after proper immune selection) with e.g. a myeloma cell. The resulting hybridoma cell then produces the wanted Mab.
  • the resulting scFv is routinely expressed in such a way that it is exported to the periplasm of the prokaryotic Gram- negative producer cell, by exploiting an excretion signal sequence.
  • other ways of producing scFvs in producer cells can be thought of.
  • E. coli the cytoplasmic expression of scFvs results in accumulation of the recombinant material in inactive form in inclusion bodies. Renaturation of these materials is possible, but high level production of active scFvs in the cytoplasm would be desirable.
  • TrxB thioredoxin reductase
  • coli functional scFv was demonstrated in the cytoplasm (Proba and Pluckthun, Gene 159: 203-207, 1995), functional expression in the cytoplasm is presumably a result of the correct formation of disulfide bridges. Functional expression in the cytoplasm is very well possible. Examples can be found with other hosts, e.g. with plants, such as tobacco, in plant or animal cell lines, yeasts, fungi, and so on. scFvs can be made by amplifying and cloning the proper region of the genes coding for a selected antibody or antibody fragment.
  • scFvs A recent break-through in the construction of scFvs has been the application of a random selection process to identify the wanted gene from a large library of genes, each coding for a particular scFv. This can be accomplished by expressing each of these scFvs on the head of a bacteriophage, in a form such that the scFv still can bind its antigen, followed by selection of phages with a high affinity for the desired antigen. This system has become known as the phage display system.
  • scFvs must also be labeled or conjugated after production with reporter molecules before they can be used as immunological reagent. Labeling can routinely be performed in many different ways: the proteins can, e.g., be labeled chemically with (fluorescent) dyes; they can be labeled by covalent attachment of a reporter enzyme; the presence of a recognizable amino acid sequence for another antibody (a 'tag') may allow the application of the so-called sandwich technique; or a radioactive label (usually a short-living radioactive cation) can be complexed to the scFv. In the latter form scFvs have been applied in the ultra-sensitive diagnostic analyses of tumor metastasis.
  • the present invention now provides a much more reproducible and quantifiable labeling procedure of specific immunological reagents with reporter molecules by in vivo labeling of a scFv or another synthetic antibody fragment by translational fusion of an antibody gene or fragment thereof with a functional reporter gene or functional fragment of a reporter gene.
  • Reporter genes or fragments thereof can for instance be selected from genes encoding reporter enzymes or fluorescent reporter proteins.
  • the resulting constructs encode fusion proteins comprising an scFv fragment specifically directed against (i.e. specifically recognizing or binding) an antigen of choice and a label by which immunochemical detection can occur.
  • the in vivo labelled scFv fragments may be used, possibly after a suitable purification procedure, as immunological reagent to detect antigen by any possible immunological detection technique.
  • the advantage of in vivo labeling of scFv fragments is that once the construct has been made via recombinant techniques, batch-to-batch or within- batch variability cannot occur; each and every time the same reactive molecule is expressed.
  • the in vivo labeling can be carried out both before (upstream of) the N-terminal part of an scFv, or after (downstream oi) the C-terminal part of an scFv, and can be combined with the attachment of additional labels or tags to an scFv.
  • the invention includes embodiments wherein the expressed product is composed of an scFv and an attachment moiety consisting of an amino acid or peptide which allows later attachment of a fluorescent chromophore.
  • the subject invention provides a recombinant nucleic acid molecule comprising at least one nucleic acid sequence encoding a variable domain, or a fragment thereof, of a heavy and/or light chain of an antibody molecule, and a nucleic acid sequence encoding a functional reporter molecule or functional fragment thereof, or encoding an attachment moiety for attachment of a reporter molecule.
  • the functional, dimeric, form is only present in the periplasm of the cell and can be found in the periplasm only at very low expression levels. In the cytoplasm no functional product is found.
  • the present invention provides a wide variety of labels or reporter molecules that do not require processes such as dimerization to render them functional. Also, the present invention provides functional immunological reagents that are produced in the cytoplasm of the host cell at high expression levels.
  • a reporter molecule such as, e.g., the ferredoxin-NADP + oxidoreductase enzyme (FNR) can be overexpressed up to levels of at least 30% of the cell weight in a soluble, active form. It is expected that the expression levels of scFv-FNR fusion proteins will roughly be equivalent to the expression levels of the unfused scFvs.
  • FNR ferredoxin-NADP + oxidoreductase enzyme
  • a class of enzymes relevant for the uses and applications of this invention are the oxidoreductases .
  • a ferredoxin-NADP + oxidoreductase enzyme from cyanobacteria has been used in the experimental part.
  • FNR also designated as PetH
  • PetH is the gene product of the petH gene; it may be isolated from the cyano- bacterium Anabaena variabilis ATCC 29413.
  • Additional oxidoreductases, that can be used in a similar way, for instance the peroxidases, like haloperoxidase from Curvularia, are also discussed as well as the substrates that can be used for their subsequent detection.
  • different classes of hydrolytic enzymes can translationally be coupled to the gene encoding the scFv.
  • ferredoxin-NADP + oxidoreductase The best studied ferredoxin-NADP + oxidoreductase is the enzyme from spinach. In its native form FNR associates with ferredoxin, a soluble Fe-S containing protein that is reduced by photosystem I during oxygenic photosynthesis. It catalyses the transfer of electrons to NADP + :
  • FNR is a flavoprotein that performs its redox reactions with a non-covalently attached FAD as the redox-active cofactor.
  • FNR has been classified within class IV, which contains the dehydrogenases/ electron transferases. In its primary structure characteristic domains can clearly be recognised: motifs that are involved in the binding of the FAD, as well as motifs that are assigned to NADP + binding.
  • the crystal structure of the FNR from spinach has been resolved (for a review see: Karplus, P.A. and Bruns, CM., 1994). This structure reveals that FNR is composed of two independently folded domains.
  • the N-terminal domain has a 'beta-barrel' structure and binds FAD.
  • the C-terminal, NADP + - binding domain is a structure consisting of beta sheets and alfa helices.
  • reductases have a similar domain structure: the FAD- NADP + domain structure can be considered as modules with widespread occurrence.
  • these homologues are also the FMN-flavoproteins and the NADH-dependent reductases.
  • Some representative examples are: Nitric oxide synthase, Cytochrome P450 reductase, Sulfite reductase, Neutrophil NADPH oxidase, Ferric reductase, Flavohemoglobin, Cytochrome b5 reductase, Nitrate reductase, Oxygenase reductases, Phthalate dioxygenase reductase, etc. These enzymes are also applicable as reporter enzyme in translational fusion constructs with scFvs.
  • the activity of FNR can be straightforwardly assayed in vitro in a reverse reaction, where oxidation of NADPH is catalysed by FNR and artificial electron acceptors such as KsFe(C ) 6 or iodonitrotetrazolium violet become reduced.
  • KsFe(C ) 6 or iodonitrotetrazolium violet become reduced.
  • K m values towards NADPH of 10-50 ⁇ M and Vmax values of 60-150 U/mg have been reported.
  • Anabaena variabilis enzyme show similar enzyme kinetics.
  • the lacZ gene encoding the ⁇ -galactosidase can be used as an alternative chromogenic tag.
  • a well known substrate for this enzyme activity is ONPG (o-nitrophenyl-beta-D-galacto- pyranoside), which is soluble in water and can be used in ELISA assays. Also the substrate chlorophenol red-beta-D- galactopyranoside can be used for ELISA detections. Fluores ein di-beta-D-galactoside is a fluorogenic enzyme substrate for galactosidase.
  • the substrate X-gal (5-bromo-4- chloro-3-indolyl-beta-D-galactopyranoside) is insoluble and can be used in western blot or immunohisto-/cytochemistry.
  • Peroxidases can be attached to scFvs.
  • water soluble substrates useful for ELISA are ABTS (2,2 '-azino-bis[3-ethyl- benzothiazoline-6-sulfonic acid], diammonium salt) and TMB ( 3, 3 ' , 5 , 5 ' -tetramethylbenzidinedichloride) and OPD (o-phenylenediaminedichloride) .
  • Known insoluble substrates are DAB (3,3' -diaminobenzidinetetrachloride) , AEC ( 3-amino-9- ethylcarbazole) and 4-chloro-l-napht ol. Also chemiluminescent substrates are commercially available.
  • Bacterial Upases and esterases are known to hydrolyze the ester bonds in the compounds p-nitrophenyl palmitate and p-nitrophenyl acetate respectively, which can be monitored in ELISA by following the extinction at 410 nm. See Kok et al . 1995.
  • GFP Green Fluorescent Protein
  • GFP can be produced in fluorescent form through heterologous expression, for instance in E. coli .
  • a posttranslational maturation process gives rise to the oxidation of one of the tyrosines of the protein, resulting in a cyclization reaction that forms an imidazolin-5-one intermediate.
  • modified forms of GFP have been selected after random mutagenesis. These (i) have a more simple and straightforward fluorescence spectrum with only a single (and stronger) absorption, and thus excitation, maximum, (ii) mature more rapidly and (iii) variants with different emission maxima are now available, so that simultaneously different variants can be detected.
  • Phycobiliproteins are an intrinsic part of the antenna system of cyanobacteria. They contain a linear tetrapyrrole chromophore that is posttranslationally attached to the apoprotein via a thiol-ether linkage. These proteins can be obtained with a range of absorption spectra, covering the visible part of the spectrum, depending on the particular structure of the tetrapyrrole chromophore and the amino acid sequence of the corresponding apoprotein.
  • Photoactive Yellow Protein is a recently discovered protein that functions as a photoreceptor in purple bacteria (Hoff, 1995). It can be heterologously produced in E. coli, be it that then only the apoprotein is formed. In vitro (and under physiological conditions) the protein can be reconstituted with a large range of chromophore analogues (A. Kroon et al . , in preparation). Among the hybrid PYP molecules thus formed are several that are highly fluorescent. The high quantum yield of this fluorescence makes these proteins also attractive tags for fluorescence detection.
  • Fluorescent proteins may also be translationally fused to the scFvs: PYP requires in vitro reconstitution, before it is fluorescent. This can be conveniently combined with cytoplasmic overexpression of the scFv fusion product, during renaturation. Also translational fusion constructs with phycobiliproteins can be constructed.
  • amino acids For the in vitro labeling of engineered scFv fragments containing an attachment moiety, use can be made of various amino acids, each with its characteristic chemical functionality. Such amino acids can be engineered at either the N-terminus or the C-terminus into the coding frame of the scFv. They can be encoded as single residue additions, or multiple additions. It should be noted that there must be a limit to the amount of added residues such as cysteine that can be introduced without interfering with either solubility or functionality.
  • Well known chemical coupling methods can be used to introduce fluorescent chromophores. One or more cysteine residues, for example, can be used for attachment of fluorescent chromophores. The chromophores can be coupled to these residues by e.g. a maleimide reaction scheme, a pyridyl reaction scheme, an active halogen reaction scheme, or via a thioester bond linkage as in the case of reconstitution of the chromophore of the Yellow Protein.
  • Tyrosine and histidine residues can be used as attachment moiety and be labeled with p-diazobenzoyl compounds.
  • Arginine residues can be coupled selectively with phenyl glyoxal compounds. Aspartate and glutamate have carboxyl groups that can be aminated with fluorescent compounds carrying primary amine groups, via a carbodiimide-activated intermediate.
  • scFv large amounts of in vivo labeled scFv can thus, according to this invention, be produced in a very simple and robust fermentation system, for example based on Gram-positive or Gram-negative bacteria and/or yeasts.
  • Production hosts could in principle be either prokaryotes or eukaryotes. In view of the high growth rate of bacteria, such as E. coli , however, these are preferable in industrial production processes.
  • various targeting signals can be used to produce these hybrid scFv-reporter protein constructs in (i) the cytoplasm, (ii) the periplasm or (iii) extracellularly.
  • the present invention provides in vivo labeled synthetic antibody fragments which can be used to detect antigens with even higher specificity and sensitivity.
  • the invention provides immunological reagents comprising specifically labeled scFv fragments that utilize an intrinsic amplification mechanism, leading to a decrease in the detection limit for rare antigens.
  • immunological reagents comprise labeled scFv fragments that specifically bind with the same receptor molecules as they are labeled with.
  • scFv fragments fused with a reporter gene can be generated that specifically recognize the reporter gene product (here the PetH enzyme is used as an example but many other enzymes can be used without altering the principle disclosed by the invention), without impairing the specific activity of the reporter gene product.
  • any scFv reacting with a particular antigen, that has translationally been fused with for instance FNR or GFP, can be reacted with scFvs that specifically bind to FNR or GFP, respectively.
  • the scFv that binds the Synechocystis PCC 6803 ferredoxin- NADP + oxidoreductase does not inhibit its enzyme activity when it is bound.
  • an antigenic determinant 'A' is bound by a scFv that is translationally fused with epitopes 'B' and 'C, it could be detected with a mixture of two scFvs -anti'B' and -anti'C that are also tagged with epitopes 'B' and 'C. In this case not a linear but an exponential amplifi- cation is achieved upon the binding of each 'next' scFv.
  • the invention thus provides immunological reagents with polyclonal activity, by preparing mixtures of in vivo labeled scFv fragments that bind different antigenic determinants. In this way a polyclonal antibody can be mimicked by using such a mixture of scFvs. Further the invention provides immunological reagents that recognise a primary antibody, by making use of a mixture of in vivo labeled scFvs. The invention also provides multivalently labeled immunological reagents by integrating more copies of the gene encoding the reporter molecule in a single overexpression construct.
  • any scFv fused to either the truncated version of the Anabaena variabilis petH gene encoding the ferredoxin NADP oxidoreductase or the gfp gene encoding the Aequora Green Fluorescent Protein, it was necessary to obtain such a fragment with binding affinity for a known antigen.
  • the sequence of the petH gene encoding the protein antigen when expressed in the cytoplasm of E. coli is available on the internet by using the 'Cyanobase' sequence retrieval programs.
  • the gene product is enzymatically very similar to the Anabaena petH fusion partner studied as the chromogenic tag, whose basic enzymology is described in this text as well.
  • the scFv 'mab4' directed against Synechocystis PCC 6803 ferredoxin-NADP + oxidoreductase is of the type VH-linker-VL, with the linker sequence (Gly 4 -Ser) 3 , a sequence which can be cloned in frame using the 5 ' Sfil and 3 ' Notl restriction sites in the cytoplasmic and periplasmic expression vectors described in this text.
  • linker sequence Gly 4 -Ser 3
  • any scFv-coding sequence that is produced with the Pharmacia recombinant phage display kit can be cloned in frame in the expression constructs described in this text, using the same 5' Sfil and 3' Notl restriction sites.
  • the expression vector sold by Pharmacia, pCANTABl is based on the pHENl vector that was constructed to clone and express the library from which (mab4) originates. Any other scFv fragment that does not have these convenient restriction sites can easily be modified for in frame cloning by a skilled worker, for example by the introduction of suitable Sfil and ⁇ Totl restriction sites by polymerase chain reaction-based mutagenesis.
  • the nucleotide sequences of the expression constructs are given in figures 2 and 3.
  • the cytoplasmic expression vectors that we have used are based on the Quiagens PQE30 series, which contain a strong T5 viral promoter that can be repressed by the overproduction of Lacig. The latter is achieved by co-replication of Lacis expressed from E. coli plasmid pREP4. Cloning of an Open Reading Frame into this polylinker results in the translational fusion of 6 histidine residues at the N-terminus of the overexpressed protein.
  • the promoter region contains two binding sites for the Lac repressor, and expression, when glucose is added to a culture, is additionally repressed.
  • the polylinker of pQE30 was replaced essentially by the polylinker of pHENI.
  • a 140 bp product generated with pHENI as the template was amplified by PCR using the oligonucleotides 'phenbam' and 'phensac'.
  • the 5' end of this polylinker fragment now incorporates a BamHI site for in-frame cloning in the expression plasmid, and the 3' end incorporates a Sad restriction site, also present in the polylinker of pQE30.
  • the PCR fragment was digested with BajziHI and Sad and cloned into the BamHI and Sad sites of pQE30.
  • the resulting plasmid, pHKI was used to generate both the ferredoxin-NADP + oxidoreductase-fused and the green fluorescent protein-fused expression vectors, both incorporating the N-terminally placed histidine-tag.
  • the purified native enzyme usually looses the N-terminal domain as well, as a result of proteolysis, and still is fully active as a reductase.
  • this truncated enzyme in periplasmic expression, it was also necessary to remove the N-terminal domain, since this domain contains a number of positively charged residues that hinder the translocation of the fusion protein over the plasma membrane.
  • the C-terminal part of the gene, encoding both the cofactor (FAD) and substrate (NADPH) binding domains was amplified by use of PCR with the oligonucleotides ' anapetkpn ' ( 5 ' ) and 'cpethind ' ( 3 ' ) .
  • This fragment was digested with Kpnl and Hindlll and cloned into the Kpnl and Hindlll sites of pHKl, to produce pHK3.
  • This vector is shown in figure 1 and accepts 'any' in frame scFv encoding sequence when cloned into it as a Sfil/Notl fragment. Without this cloning procedure induction of an E. coli strain harbouring this plasmid leads to the cytoplasmic over- expression of the N-terminally truncated reductase, fused at the N-terminus with respectively a histidine tag, a short nonsense peptide encoded by the polylinker, and an epitope- coding sequence called 'c-myc'.
  • This epitope sequence could function as a 'linker' between the scFv and the reductase, but is meant to study the expression of the chimeric proteins when detected in western blot by the mouse monoclonal antibody 9E10.
  • an scFv coding sequence is introduced into this plasmid, it will replace the nonsense sequence in pHK3. This was done with the DNA fragment coding for the scFv directed against Synechocystis ferredoxin-NADP + oxidoreductase 'mab4', resulting in construct pHK4.
  • the tag- and domain structure of the chimeric protein would than become (from N- to C-terminus): (His) 6 -VH-linker- VL-myc-FAD-NADPH.
  • the order of these domains can also be selected in a very different way, like the reductase being placed N-terminally of the scFv fragment, or on any other side.
  • scFvs with a reversed order of appearance of variable genes: VL-linker-VH have been successfully produced.
  • domains can be left out at will, for instance, constructs without for example VI and/or myc domains also encode functional immmunological reagents.
  • a vector, pHK6, was generated based on pHK3, to accept a Sfil/Notl scFv encoding fragment for the expression and trans- location of the scFv-reductase fusion protein to the periplasm. To this end three fragments were ligated to produce pHK6.
  • the first DNA fragment contains the polylinker and the petH ' sequence and was isolated as a 1 kbp Sfil /HindiII fragment.
  • the second DNA fragment was generated by PCR with the vector pHENI as template, using the primers 'phenbam' and 'phensac'. This fragment was also restricted with Sfil and Hindlll and contains the leader sequence 'PelB'.
  • the lac promoter and the translational signals were provided by pUC19, that was digested with HindiII to accept the other two fragments.
  • pHK6 the 'mab4 ' coding sequence was cloned to produce pHK7, a high copy number plasmid (due to the use of pUC19) that overexpresses a chimeric protein with a domain structure PelB-VH-linker-VL- myc-FAD-NADPH.
  • the PelB leader is cleaved off by the enzyme • signal peptidase' after translocation of the construct over the cytoplasmic membrane by the secretory machinery (Sec) of E. coli, resulting in a domain structure VH-linker-VL-myc-FAD- NADPH.
  • This protein accumulates in the periplasm and can be isolated by affinity chromatography with Red Sepharose from the periplasmic cell fraction.
  • the gfp gene was cloned into the polylinker of pHKI as a Kpnl/Smal fragment.
  • the gfp containing plasmid pGFP was purchased from Clonetech.
  • An 0.7 kbp .Kpnl/Hindi11 fragment encoding gfp was first cloned into pBluescript-II KS+, to add the suitable Smal restriction site at the 3' end of the gene, in order to clone it directionally into pHKI, which resulted in pHK2.
  • the (mab4) coding fragment was cloned as a Sfil/Notl fragment into this construct to produce pHK5.
  • the resulting domain structure of this fusion protein is (His) 6 -VH-linker-VL-myc-GFP. Since it has been shown that GFP can also be fused as an N-terminal fusion partner, a chimeric product with a reversed domain structure, or with the GFP on either side of the scFv fragment, could also be expressed.
  • a vector for the periplasmic expression of the scFv-GFP was based on pHK2.
  • the polylinker sequence and the gfp coding region was excised from pHK2 as a 0.8 kbp Sfil/EcoRI fragment and ligated to the 3.2 kbp Sfil/Ec ⁇ RI fragment of pHENI, resulting in pHK8.
  • the ' mab4 ' coding fragment was cloned as a Sfil/Notl fragment into this construct to produce pHK9.
  • This construct is a high copy number plasmid (based on pUC119) that overexpresses a chimeric protein with a domain structure PelB- VH-linker-VL-myc-GFP, that looses its N-terminal PelB leader sequence after translocation to the periplasm.
  • Phase contrast microscopy (not shown) showed the formation of inclusion bodies inside the cytoplasm of both cultures upon induction, indicating that the overexpressed fusion protein precipates in the cytoplasm. Also, a slight elongation of the cell morphology was observed, which is not uncommon as a result of overexpression conditions.
  • the chimeric protein was visualised in SDS-PAGE by staining with Coomassie Blue (total protein stain) and by immunoblots (Western blots) with rabbit-anti-Ana&aena variabilis ferredoxin-NADP + oxidoreductase antibody, which exclusively stains the fusion protein.
  • the results are shown in figures 4 and 5.
  • the arrow indicates the position of the protein in both the Coomassie stained gel and in the immunoblot.
  • an additional band with the correct molecular mass is observed only in strain BMH71-18.
  • the immunostaining shows the overexpression in both strains.
  • E. coli strains Three E. coli strains, XLI-Blue, M15 and BMH71-18, were tested for their capacity to express the scFv-fusion construct mab4 in their periplasm. Plasmid pHK7, encoding the PelB-VH- linker-VL-myc-FAD-NADPH chimeric protein, was originally constructed in E. coli XLI-Blue. Strains that were analysed to harbour the correct construct after ligation and tranformation had a clear phenotype, especially in liquid medium, even when additional glucose was supplied to the cultures to repress 'leaky' expression from the lac-promoter. Also an exceptional filamentous cell morphology was observed, indicative of the presence of a toxic gene construct.
  • the purpose of this experiment was to show the functionality of the fusion protein by using soluble material from the periplasm. As expected the expression levels are lower than the cytoplasmic expression levels, but can be used directly to study the functionality. These expression levels of native protein are limited to the volume of the periplasm, and native expression levels could be increased by overexpressing the fusion proteins in Gram-negative bacteria such as Acinetobacter sp. or Pseudomonas sp. that are able to actively transport proteins over the outer membrane as well, resulting in the accumulation of material in the extracellular supernatant. For this purpose motifs that are recognised by the Xcp machinery should be added to the constructs.
  • periplasmic fraction was performed with cells harvested from 1 liter of induced culture of BMH71- 18 (pHK7) (pREP4). Proteins were precipitated from the resulting preparation by the addition of 80% of ammonium sulfate. The pellet was taken up in a buffer containing 50 mM Tris pH 8 + 50 mM NaCl + 1 ⁇ M FAD and desalted on a Sephadex G 50 column. Fractions that were visibly yellow, as a result of binding of FAD by the flavoprotein, were stored at -20°C after addition of 30% of glycerol. This procedure results in the preparation of a concentrated extract of the periplasmic proteins, where the FNR activity i ⁇ reconstituted by the addition of free FAD.
  • the presence of the fusion protein could be detected in ELISA in which the coated antigen was detected by the fusion protein, and it was shown that the fusion protein was able to detect the antigen in Western blot, by means of its diaphorase activity, employing the reaction mixture described below.
  • the scFv-FNR fusion protein can be purified on a Red Sepharose column.
  • the results are shown in figure 7. This chromatographic procedure is based on the affinity that NAD(P)H dependent enzymes have for this column material. After extraction of the periplasmic proteins they were dialysed in the presence of 0.5 mM of PMSF against a glycine buffer (50 mM glycine, 50 mM KC1). A 1 ml Red Sepharose was equilibrated with the glycine buffer. Proteins were loaded on the column, washed with glycine buffer, and eluted with a gradient of 0-1M of KC1 in glycine buffer. Fraction 3 contained the purified scFv-FNR fusion protein, as visualised by SDS-PAGE and assaying the diaphorase activity.
  • the diaphorase activity of the enzyme can be detected by the reduction of tetrazolium salts.
  • tetrazolium salts Several of these salts were tested for their ability to be reduced by the reductase. Tetrazolium blue, nitrotetrazolium blue and iodotetrazolium violet all become visibly colored (blue, blue and violet, respectively) upon reduction, whereas the oxidized materials are virtually colorless or slightly yellowish. Of these three, iodotetrazolium blue (INT) was more readily reduced by the reductase than the other three. For the detection of the enzyme activity, both in ELISA and in Western blot, INT is reduced by electrons liberated from NADPH, catalysed by the reductase.
  • NADP + can be re-reduced by the enzyme glucose-6-phosphate dehydro- genase (G-6-P DH), which is an NADP+ oxidoreductase. If G-6-P DH and G-6-P are added to the reaction mixture the components do not become exhausted, and replace the requirement of high concentrations of NADPH in the reaction mixture.
  • G-6-P DH glucose-6-phosphate dehydro- genase
  • Tris.HCl pH 8 1 mM NADPH 2 mM INT (from stock solutions in dimethyl formamide) 10 mM Glucose-6-phosphate 5 U/ml Glucose-6-phosphate dehydrogenase (from Boehringer).
  • Fluorescent proteins can be translationally fused to scFvs: Good examples are green fluorescent proteins (GFP) from Aequorea victoria and Renyella and Photoactive yellow protein (PYP) from Ectothiorhodospira halophila . Recently the gene encoding the E. halophila Photoactive Yellow Protein has been cloned and sequenced (Hoff, W. 1995). Studies of reconstitution of the chromophore on the over- expressed recombinant protein showed that fluorescent analogs of the p-coumaric acid can be linked to the apo-protein to produce a fluorescent holo-protein. These in vitro reconstitutions are specific for the unique cysteine residue in the protein.
  • the reconstitution can be carried out by reacting the functional fragment apoform of the protein with the anhydride derivatives of these chromophores. These same couplings can therefore be performed by fusing one or more cysteine residues to the scFv coding sequence and performing the reconstitution in vitro with the desired chromophores.
  • the colors as well as the fluorescent intensities can be varied in this way by choosing the correct coumarin analogue and the amount of cysteine residues to be coupled.
  • the analogues studied in the photoactive yellow protein environment and their fluorescence properties are listed below. Compound Extinction ⁇ Max (nm) ⁇ Em (nm) (mM- 1 .cm -1 )
  • An other example of an 'activated' coumarin analogue is N-[6-(7-amino-4-methylcoumarin-3-acetamido)hexyl]-3 '-(2 '- pyridyldithio)propionamide (AMCA-HPDP).
  • AMCA-HPDP N-[6-(7-amino-4-methylcoumarin-3-acetamido)hexyl]-3 '-(2 '- pyridyldithio)propionamide
  • This compound is sold by Pierce Chem. Comp.
  • the resulting fluorophore absorbs at 345 nm and emits at 440-460 nm and has a high quantum yield and photostability.
  • translational fusion constructs with phycobili- proteins can be constructed.
  • Phycoerythrin (Absorption 450- 470 nm, Emission 574 nm) has already been used as a fluorescent marker by many researchers, and is sold as purified or conjugated isolate by companies such as Pierce. Depending on the source of isolation, phycoerythrin mostly contains the phycoerythrobilin chromophore, but can also contain phycourobilin. The chromophores are attached via one or two thioether linkages. This opens the possibility to covalently couple these chromophores via a thioether linkage to cysteine residues that are engineered as functional fragment into the coding frame of a scFv fragment. Figure legends
  • Figure 1 Physical maps of the cytoplasmic and periplasmic expression vectors of the FNR and GFP fusion constructs.
  • Figure 2 Sequence of the coding region of the cytoplasmic FNR fusion expression vector. Positions of motifs and restriction sites are given relative to the initiation codon.
  • Figure 3 Sequence of the coding region of the periplasmic FNR fusion expression vector. Positions of motifs and restriction sites are given relative to the initiation codon.
  • Figure 4 Coomassie stained SDS-PAGE of total E. coli proteins. Lane 1: BMH 71-18 (pREP4) (pHK4), induced with IPTG. Lane 2: BMH 71-18 (contruct free). Lane 3:Ml5 (pREP4) (pHK4), induced with IPTG. Lane 4: M15 (construct free). The arrow indicates the position of the (visible only in lane 4) scFv- FNR fusion protein.
  • Figure 5 Western detection of the same SDS-PAGE gel shown in figure 4 using a rabbit anti Anabaena variabilis FNR polyclonal antiserum. The arrow indicates the position of the scFv-FNR fusion protein.
  • Figure 6 Western detection of the periplasmic scFv-FNR fusion protein.
  • Lane 1 Total proteins E. coli BMH 71-18 (pHK7), induced with IPTG. The upper arrow indicates the position of the PelB-scFv-FNR fusion protein not yet cleaved by the periplasmic signal peptidase. The lower arrow indicates the position of the cleaved, periplasmic, scFv-FNR fusion protein.
  • Lane 2 Periplasmic extract from induced BMH 71-18 (pHK7). The arrow indicates the position of the cleaved, soluble, periplasmic scFv-FNR fusion protein.
  • Figure 7 Coomassie stained SDS-PAGE. Purification of the soluble periplasmic scFv-FNR fusion protein on Red Sepharose. Lane 1: BMH 71-18 (construct free). Lane 2: Total proteins E.coli BMH 71-18 (pHK7), induced with IPTG. Lane 3: Periplasmic extract from induced BMH 71-18 (pHK7). Lane 4: Fraction 2. Lane 3: Fraction 3. The arrow indicates the position of the purified protein. References

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Abstract

Cette invention concerne des fragments d'anticorps à chaîne unique, étiquetés et in vivo qui sont obtenus par l'expression dans une cellule hôte d'un acide nucléique recombinant comprenant les éléments suivants: une partie codant un domaine variable d'une chaîne lourde et/ou légère d'une molécule d'anticorps; et une partie codant un ligand fonctionnel, tel qu'une enzyme ou une protéine fluorescente, ou un fragment de fixation permettant de fixer un chromophore fluorescent. Ces fragments d'anticorps à chaîne unique, étiquetés et in vivo peuvent être utilisés en qualité de réactifs immunologiques.
PCT/NL1997/000653 1996-11-28 1997-11-27 PRODUCTION DE FRAGMENTS D'ANTICORPS SYNTHETIQUES, A CHAINE UNIQUE, ETIQUETES ET $i(IN VIVO) WO1998023737A1 (fr)

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FR2778408A1 (fr) * 1998-05-11 1999-11-12 Univ Pasteur Anticorps recombinants fluorescents
EP1240314A1 (fr) * 1999-12-21 2002-09-18 The Regents of the University of California Genes chimeres fluorescents a base de phycobiliproteine recombinee multifonctionnelle et presentation de phycobilisome
WO2003093321A1 (fr) * 2002-05-06 2003-11-13 Affibody Ab Polypeptide de fusion, utilisation de ce polypeptide de fusion et methodes d'emploi de ce dernier
WO2010111299A2 (fr) * 2009-03-24 2010-09-30 The Arizona Board Of Regents A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizona State University Anticorps de synthèse
CN105567621A (zh) * 2016-02-03 2016-05-11 武汉科技大学 一种促进细胞内辅酶nadph再生的基因工程集胞藻及其应用

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2778408A1 (fr) * 1998-05-11 1999-11-12 Univ Pasteur Anticorps recombinants fluorescents
EP1240314A1 (fr) * 1999-12-21 2002-09-18 The Regents of the University of California Genes chimeres fluorescents a base de phycobiliproteine recombinee multifonctionnelle et presentation de phycobilisome
EP1240314A4 (fr) * 1999-12-21 2003-02-26 Univ California Genes chimeres fluorescents a base de phycobiliproteine recombinee multifonctionnelle et presentation de phycobilisome
US6649376B1 (en) 1999-12-21 2003-11-18 The Regents Of The University Of California Multifunctional recombinant phycobiliprotein-based fluorescent constructs and phycobilisome display
US7169582B2 (en) 1999-12-21 2007-01-30 Regents Of The University Of California Multifunctional recombinant phycobiliprotein-based fluorescent constructs and phycobilisome display
US7176000B2 (en) 1999-12-21 2007-02-13 Regents Of The University Of California Multifunctional recombinant phycobiliprotein-based fluorescent constructs and phycobilisome display
WO2003093321A1 (fr) * 2002-05-06 2003-11-13 Affibody Ab Polypeptide de fusion, utilisation de ce polypeptide de fusion et methodes d'emploi de ce dernier
WO2010111299A2 (fr) * 2009-03-24 2010-09-30 The Arizona Board Of Regents A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizona State University Anticorps de synthèse
WO2010111299A3 (fr) * 2009-03-24 2011-03-31 The Arizona Board Of Regents A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizona State University Anticorps de synthèse
CN105567621A (zh) * 2016-02-03 2016-05-11 武汉科技大学 一种促进细胞内辅酶nadph再生的基因工程集胞藻及其应用
CN105567621B (zh) * 2016-02-03 2019-10-11 武汉科技大学 一种促进细胞内辅酶nadph再生的基因工程集胞藻及其应用

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