WO1995006125A1 - RECEPTEUR CHIMERIQUE CONTENANT UN DOMAINE DE FIXATION DE L'IgG COMMUN AUX PROTEINES A ET G - Google Patents

RECEPTEUR CHIMERIQUE CONTENANT UN DOMAINE DE FIXATION DE L'IgG COMMUN AUX PROTEINES A ET G Download PDF

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Publication number
WO1995006125A1
WO1995006125A1 PCT/US1994/009141 US9409141W WO9506125A1 WO 1995006125 A1 WO1995006125 A1 WO 1995006125A1 US 9409141 W US9409141 W US 9409141W WO 9506125 A1 WO9506125 A1 WO 9506125A1
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peptide
protein
binding
region
amino acid
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PCT/US1994/009141
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English (en)
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Young Moo Lee
Sohel Talib
Thomas B. Okarma
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Applied Immune Sciences, Inc.
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Priority to AU75635/94A priority Critical patent/AU7563594A/en
Publication of WO1995006125A1 publication Critical patent/WO1995006125A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to novel polypeptide compositions having Fc receptor activity, methods for use of the polypeptides, and methods for their preparation using recombinant DNA techniques.
  • Fc receptors are unique proteins found on the surface of certain bacteria which bind selectively to the constant Fc region of IgG from many mammals by a non- immune mechanism.
  • Protein A is the type I Fc receptor from Staphylococcus aureus .
  • SpA is structurally and functionally pentavalent in IgG binding (Moks, et al., Eur. J. Biochem. (1986) 156:637-43).
  • protein A consists of five homologous binding domains (E, D, A, B, & C) , each of which is capable of binding to the Fc region of IgG.
  • the deduced amino acid sequence of protein A reveals extensive sequence homology between the five domains (a 58 amino acid unit per domain) .
  • Protein G is a cell surface protein of group G Streptococci . It is analogous to protein A. Protein G (SpG) is structurally and functionally trivalent in its IgG-binding ability, protein G consists of three homologous IgG binding domains (CI, C2 and C3) . Protein G binds to several animal IgGs and IgG subclasses, including human IgG3, to which protein A does not bind. Amino acid sequence analysis reveals that the three IgG binding domains (CI, C2, C3) are well conserved, particularly between CI and C2. The minimal size of an IgG binding seems to be 55 amino acid units (each C domain) . (Guss, et al., EMBO J. (1986) 5_:1567-1575) Unlike protein A, no X- ray crystal structure of Fc-protein G complex is available.
  • Staphylococcal protein A and Streptococcal protein G both exhibit high affinity for the Fc portion of immunoglobulin G (IgG) from many mammalian species.
  • IgG immunoglobulin G
  • This property of SpA and SpG has been exploited extensively in qualitative and quantitative immunology.
  • these proteins have immune therapeutic applications, such as extracorporeal plasmapheresis for the treatment of certain cancers, autoimmune diseases, and other immune abnormalities.
  • SUBS ⁇ UTE SHEET (RULE 2® It would therefore be of interest to combine the IgG binding domains of protein G and protein A in a single protein. In addition, it would be of interest to analyze the structure of protein A and protein G and to determine the relationship between structure and function of these unique receptors.
  • Protein A-protein G chimeric receptors have been reported previously (Eliasson et al., J. Biol. Chem. (1988) 263:4323-4327; Sun and Lew, J. Immunol. Methods (1992) 151:43-28).
  • Protein A-protein G chimeric Fc receptors have complementary binding patterns, and retain the binding capacities of both the parental constituents A and G (Eliasson et al., J. Biol. Chem. (1988) .262:4323-4327; Sun and Lew, J. Immunol. Methods (1992) .152:43-28).
  • the chimeric proteins of Eliasson et al. either contained the whole protein A (5 IgG binding domains) and the entire protein G (3 IgG binding domains) , or two IgG binding domains of protein A and the entire protein G.
  • Sun et al. constructed their tripartite protein by combining whole protein A, two IgG domains of protein G and glutathione S-transferase.
  • novel peptide compositions which comprise binding domains and fragments thereof of protein A and/or protein G; also provided are chimeric peptides comprising at least one binding domain from each protein.
  • the construct of the present invention is the smallest chimer possible, consisting of only one domain from each of two different
  • SUBSTITUTI SHEET (RULE 26) receptors SpA and SpG.
  • This chimer has no domain redundancy yet still maintains biological activity, in contrast to all other chimeric molecules that have multiple domains from separate proteins.
  • the chimeric peptides can be obtained by recombinant means, wherein an expression vector comprising a DNA sequence encoding an Fc receptor or a biologically active fragment thereof is used to transform host cells. The transformed cells are then grown under conditions whereby the DNA sequence is expressed and the desired expression product is obtained.
  • the compositions are characterized as comprising at least one Fc receptor retaining SpA and/or SpG specificities and capable of binding specifically to an immunoglobulin G (IgG) molecule.
  • the compositions find use, for example, in detecting the presence and quantity of IgG in a biological fluid test samples.
  • Figure 1 is a schematic drawing of the structure of protein A and protein G, and gene cassettes used to construct synthetic protein G (SG) (constructed with plasmid pMDl, containing gene MD1) and a chimeric protein SBG (constructed with plasmid pMD2, containing gene MD2) .
  • the pSGl and pSG2 plasmids code for IgG binding domains C2 and C3, respectively;
  • pSG3 codes for the proline rich hydrophilic W region;
  • pSB codes for one of the IgG binding domains (B) of staphylococcal protein A.
  • Figure 2A-D shows the nucleotide sequence of the synthetic DNA fragments used to construct SG-1 (Fig. 2A) , SG-2 (Fig. 2B) , SG-3 (Fig. 2C) , and SB (Fig. 2D) .
  • Numbers in parenthesis identify the oligonucleotides used in gene assembly. The lines denote the ends of the oligos and the respective overlapping regions between fragments. Unique restriction sites are shown.
  • Figure 3 shows the strategy of cloning synthetic protein G (via plasmid pMDl) and chimeric protein BG (SBG) (via plasmid pMD2) .
  • Gene cassettes SG-1, SG-2, SG-3 and SB were individually cloned.
  • Intermediate cassette SG-2 + SG-3 was obtained by fusion of SG-2 and SG-3 in plasmid pKK 233-2.
  • Final plasmids pMDl and pMD2 were obtained by fusion of gene SG-1 or SB into pKK SG-2 + SG-3, respectively.
  • Gene segments and plasmids have not been drawn to scale.
  • Restriction endonuclease abbreviations are as follows: Bl, BamH-1; Nl, Ncol; PI, Pstl; Kl, Kpnl; H3, Hind III; other abbreviations include pUC-SGl, pUCl ⁇ synthetic protein Gl (SG-1); pUC-SG2, pUC19 synthetic protein G2 (SG-2); pUC-SG3, pUC19 synthetic protein G3 (SB-3) ; pKK-SB, pKK 233-2 synthetic B fragment (SB).
  • Figure 4A-B Fig. 4A shows the nucleotide and inferred amino acid sequence of the synthetic protein G gene (MD1) .
  • N-terminal factor Xa cleavable linker, IgG binding domains C2, C3 and C-terminal hydrophilic region W are shown by arrowheaded lines on the top strand.
  • Three different gene segments comprising the MD1 gene SG-1, SG-2 and SG-3, are indicated by arrowheaded lines on the bottom strand.
  • the junction sequence with pKK 233-2 and termination codon in the plasmid is also indicated.
  • Asterisks represents Asn to Gin change, to delete internal hydrazine cleavable site.
  • Figure 4B shows the nucleotide and inferred amino acid sequence of the chimeric protein SBG gene (MD2) contained in plasmid pMD2.
  • MD2 gene chimeric protein SBG gene
  • Three segments comprising the MD2 gene, B, SG-2 and SG-3 are indicated by arrowheaded lines.
  • the N-terminal Factor Xa cleavable linker sequence, C-terminal pKK 233 junction sequence and termination codon are also indicated.
  • Asterisks denote Asn to Gin substitutions.
  • Figure 5A-B shows the physical maps of expression vectors pMDl (Fig. 5A) and pMD2 (Fig. 5B) .
  • the vectors were constructed by insertion of the synthetic protein G gene (MD1) or the chimeric protein SBG gene (MD2) into the Ncol and Hind III sites of the pKK 233-2 expression vector as described herein. Expression of the genes is under the control of the tac promoter. The presence and orientation of the tac promoter (Ptac) , G and BG gene and ampicillin resistance gene (Amp) are indicated.
  • MD1 synthetic protein G gene
  • MD2 chimeric protein SBG gene
  • Figure 6 shows the results of expression of synthetic protein G and chimeric BG genes in E. coli .
  • the cultures of JM107 harboring pMDl or pMD2 were grown in LB and induced as described herein. The products were analyzed by SDS-PAGE. Lane H l":uninduced JM107 containing pMDl; Lanes 2 and 3:induced pMDl; Lanes 4 and 5 induced pMD2.
  • Figure 7 shows the immunoblotting of synthetic protein G and chimeric BG. E. coli expressed protein G and BG were purified as described herein. Protein samples were separated by SDS-PAGE, transferred onto nitrocellulose, incubated with human IgG and diluted with F(ab') 2 goat ⁇ human HRP conjugate. Lanes 1 and 2: chimeric protein BG; Lanes 3 and 4: synthetic protein G; and Lane 5: protein A standard.
  • Figure 8 shows comparative binding of human IgG subclasses as the relative amount of a given human immunoglobulin subclass required to obtain 50% inhibition of rabbit polyclonal IgG towards recombinant receptor SBG, SG, rPA (recombinant wild-type protein A) and rPG (recombinant wild-type protein G) . All numbers have been standardized so that unconjugated Rabbit IgG competes at a 1:1 level.
  • Figure 9 represents the B fragment, which is the B region of protein A.
  • Figure 10 represents C2D2, which is the C2 region of protein G with a portion of the D2 unit, as indicated in Figure 2A.
  • Figure 11 represents the Fc receptor activity of synthetic peptides and natural protein G.
  • the synthetic peptides (1 to 30 mM) or protein G (300 nM to 1 ⁇ M) in 0.05 M carbonate buffer, pH 9.3, was immobilized on a microtiter plate by overnight incubation. The plate was then incubated with 100 ⁇ l of human IgG in 0.25% BSA, 0.05% Tween-20 for 2 hours. After thorough washing of the plate, the plate was placed with the peroxidase conjugated goat anti-human IgG and incubated for 1 hour.
  • substrate solution (4.6 mM 0- phenylenediamine and 0.006% H 2 0 2 ) was placed in each well and the reaction proceeded for one-half hour.
  • the plate was read at 490 nm after sulfuric acid fixation.
  • Figure 12 represents the measurement of IgG binding activity of the synthetic peptide and the natural protein G.
  • the plate was coated with the synthetic C2D2 peptide or protein G followed by incubation with human IgG as described in Fig. 11.
  • the plate was placed with nonconjugated goat anti-human IgG and incubated for 2 hours. After washing the plate, the acid phosphatase conjugated rabbit anti-goat IgG was placed in each well of the plate and incubated for 1 hour. After washing the plate, substrate solution (0-nitrophenyl phosphate) was placed. The reaction was monitored at 405 nm.
  • Figure 13 represents the measurement of IgG binding activity for the covalently attached C2D2 synthetic peptide or the natural protein G for the sulfonium plate.
  • the peptide or protein in PBS was covalently attached to the bromoacetate by incubation overnight at 30°C. The rest of the procedure is the same as described in Fig. 12.
  • Figure 14 represents the measurement of IgG binding activity of the covalently attached C2D2 peptide to the bromoacetate plate.
  • the peptidase in 0.1 M borate buffer was placed in a covalent attachment plate and incubated overnight at 30°C. The rest of the procedure is the same as described in Fig. 12.
  • compositions comprising Fc receptors, synthetic Fc receptors, and methods of making and using these receptors are described. These compositions retain the IgG-binding capability of natural Fc receptor molecules with which they have some natural homology. Thus, the receptors bind to the constant region of the heavy chain of IgG, without influencing the ability of the IgG to recognize its antigens.
  • the compositions include chimeric receptors which have dual binding capabilities that offer the advantage that they can be used to analyze the structure and function of Fc receptors.
  • the compositions of the invention have a variety of applications including use in cell culture, and in diagnostics, and as immunogens in the production of antibodies. Polynucleotide sequences may be isolated or prepared which may be introduced into an expression vector f 0r expression of the subject compositions.
  • compositions comprise peptides which have homology in Fc receptors and biologically active fragments thereof, preferably without domain redundancy.
  • Fc receptor peptides include Protein A Ig-binding domains A-E.
  • CI, C2 or C3 domains of Protein G are alternate choices, since they comprise the Ig binding domain, and are the smallest units, which is one binding domain.
  • compositions are characterized as capable of specifically binding the constant region on the heavy chain of IgG substantially the same as natural Fc receptor molecules.
  • capable of specifically binding is intended both covalent and noncovalent binding, so that a natural ligand such as Protein A or Protein G is displaced or inhibited from binding to the synthetic Fc receptor.
  • the composition will generally have a dissociation constant (Kd) of at least about 10" 7 M, preferably at least about 10 " 9 M, more preferably at least about 10" 10 M.
  • the composition will be either an agonist or an antagonist of a natural ligand although the composition may also be a partial agonist or antagonist, where binding of a natural ligand is only partially blocked or displaced, generally by at least about 30%.
  • An important aspect in studying the relationship between the structure and function of protein is to link the roles of the individual side chains of the amino acids of proteins to the chemical, physical, and biological properties of proteins.
  • One of the most efficient techniques in studying this type of research is site-specific mutagenesis, systematically replacing an amino acid under study, with another.
  • the chimeric molecule of the invention is advantageous for use in carrying out structure-function relationship studies of Fc receptors by virtue of its small size.
  • the chimeric molecule does not have domain redundancy, unlike the natural protein A or G.
  • the chimeric molecule provides the most efficient way of systematically substituting amino acids to facilitate research.
  • Were replacement of amino acids in protein A carried out one would be required to replace the amino acid in four different domains (or 3 domains in protein G) compared with just one domain in the receptor.
  • the chimeric molecule can be loaded on the solid surface at higher density than protein A or G, perhaps by virtue of its smaller size and by inclusion of the W region (Fig. 1) .
  • the W region is comprised of two segments. One is a proline-rich domain which is located in the midst of the immunoglobulin binding region, the other segment is lysine-rich.
  • the proline-rich segment is predicted as a ⁇ -turn structure (poly proline conformation) , so that this region can be flexible and separate the C region from the lysine-rich region (repeats of Asp-Asp-Ala-Lys-Lys) . Lysine residues will be used for covalent attachment of the receptor to the membrane.
  • the chimeric molecule is more resistant to denaturation and can be bound in a uniform orientation on a solid surface by virtue of having the W region (Fig. 1) .
  • Non-native polypeptides can be produced by a number of methods.
  • oligopeptides containing neutralizing epitopes can be prepared synthetically by known techniques. See, e . g. , U.S. Patent No. 4,735,896. It is preferred, however, to prepare the non-native peptide by recombinant DNA methods.
  • the subject compositions can be prepared in a variety of ways, depending on the size of the composition. Particularly below about 80, and more particularly below about 60 amino acids, the composition can be prepared by synthesis in accordance with conventional methods. See, for example, Barany and Merrifield, "Solid-Phase Peptide Synthesis," in The Peptides: Analysis. Synthesis. Biology. Vol. 2. Special Methods in Peptide Synthesis. Part A. pp. 1-284 (Gross and Meienhofer, eds.. Academic Press, New York, 1980); see also, U.S. Patent No. 4,127,526.
  • hybrid DNA technology can be employed, where DNA sequences can be used which code for the designed polypeptide or precursor thereof.
  • DNA sequences encoding an Fc receptor or fragments thereof can be synthesized employing conventional techniques such as overlapping single strands which may be ligated together to define the desired coding sequence.
  • the termini can be designed to provide restriction sites, or one or both termini may be blunt ended for ligation to complementary ends of an expression vector.
  • an initial methionine is provided for expression of the sequence.
  • Expression vectors are generally available and they are amply described in the literature.
  • the gene may be isolated by various techniques. For example, mRNA can be isolated from a cell which codes for the Fc receptor, the mRNA reverse transcribed, the resulting single stranded (ss) DNA used as a template to prepare double stranded (ds) DNA, and the ds DNA isolated.
  • Another technique is to isolate a piece of the genomic DNA, using an appropriately degenerate probe comprising a region with the most conserved sequences in the Fc receptor gene, to thereby identify sequences encoding Fc receptor in the cell genome.
  • the probe can be considerably shorter than the entire sequence, but should be at least 10, preferably at least 14, more preferable at least 20 nucleotides in length. Longer oligonucleotides are also useful, up to the full length of the Fc receptor gene. Both DNA and RNA probes can be used.
  • the probes are typically labeled in a detectable manner (for example, with 32 p- or biotinylated nucleotides and are incubated with ss DNA or RNA from the cell in which a gene is being sought. Hybridization is detected by means of the label after ss and ds
  • oligonucleotide refers to both labeled and unlabeled forms.
  • restriction sites may be designed into the DNA encoding this polypeptide.
  • the restriction site(s) is silent with respect to the amino acid sequence, i.e., it leaves the amino acid sequence of the polypeptide unaltered, for example, changing a serine codon from AGC to AGT or an isoleucine codon from ATT to ATC.
  • incorporation of the new restriction site(s) may yield an altered amino acid sequence without substantially changing the activity of the protein.
  • Recombinant polypeptides are preferably produced according to the present invention by constructing an expression cassette and transforming a host cell therewith. Thereby, a cell line or culture is provided that is capable of expressing the relevant portion of the gene antigen which is encoded within the expression cassette.
  • the first step in constructing the expression cassette is to obtain a coding sequence of interest.
  • Encoding sequences can either be prepared directly by synthetic methods based on the disclosed sequence (or equivalent sequences encoding the same amino acids) , or by using the disclosed sequence to design oligonucleotide probes to clone coding sequence using known techniques. See, e.g., Mayfield, et al., J. Virol.. (1983) 42:259-264.
  • Synthetic coding sequences will also allow for the convenient construction of coding sequences which express analogs or "muteins".
  • coding sequences for muteins can be prepared by site-directed mutagenesis of native nucleotide sequences. The techniques of site-directed mutagenesis are known in the art.
  • the coding sequence, as described above, for the relevant portion of the gene is then operably linked to control sequences (e.g., a promoter, etc.), so that the DNA sequence encoding the relevant gene portion is transcribed into messenger RNA in a host cell transformed by the expression cassette.
  • control sequences e.g., a promoter, etc.
  • the subunit antigen coding sequence to appropriate control sequences in order to bring about expression of the desired polypeptide. See also: Harpold et al., Nucl. Acids Res. (1978) .5:239; March et al., Nature (1985) 315:641; and Parnes, J.R. et al., Proc. Natl. Acad. Sci.
  • Coding sequences for SGI, SG2, SG3 and SB are shown in Figures 2A-2D.
  • the coding sequence can be comprised entirely of the Fc encoding sequences, or such sequences can be fused to other sequences (e.g., leader sequences) so that a fusion protein is encoded. See, e.g., U.S. Patent Nos. 5 4,431,739; 4,425,437; 4,338,397.
  • the vector is capable of replication to a relatively high copy number in bacteria, since the cloning vectors have an efficient replication system functional in the host 0 bacterium.
  • the cloning vector will have at least one unique restriction site, usually a plurality of unique restriction sites and may also include multiple restriction sites.
  • the cloning vector will 5 have one or more markers which provide for selection of transformants.
  • the markers will normally provide resistance to cytotoxic agents such as antibiotics, heavy metals, toxins and the like; complementation of an auxotrophic host; or immunity to phage.
  • procaryotic expression vectors are 5 known in the art. See, e.g., U.S. Patent Nos. 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246; 4,342,832; see also U.K. Publication Nos. GB2,121,054; GB2,008,123; GB2,007,675; and European Publication No. 103,395.
  • the preferred procaryotic expression vectors are those for E. coli .
  • a number of vectors are readily available for cloning in gram-negative bacteria, especially E.
  • coli including such 5 vectors as pBR322, pUC18, pUC19, SP6, and the like.
  • Other preferred expression vectors are those for use in eucaryotic systems.
  • Yeast expression vectors are also known in the art. See, e.g., U.S. Patent Nos. 4,446,235; 4,443,539; 4,430,428; see also European Publication Nos. ° 103,409; 100,561; 96,491.
  • Examples of recombinant DNA vectors for cloning (and host cells which can be transformed) include: various bacteriophage lambda vectors (E. coli) , pBR322 (E. coli ) , pACYC171 (E. coli) , pKT230 (gram-negative 5 bacteria) , pGV1106 (gram-negative bacteria) , pLAFRl (gram- negative bacteria) , pME290 (non-J?. coli gram-negative bacteria) , pHV14 (E.
  • SV40 early region promoter SV40 early region promoter
  • Rous sarcoma virus LTR promoter
  • etc. Various mammalian expression vectors employing viral promoters (e.g., SV40 early region promoter, Rous sarcoma virus, LTR promoter, etc.) are also 0 well known in the art. See, e.g., Pachl et al. (1987), J. Virol. .61:315-325; Gorman et al. (1982) Proc. Natl. Acad. Sci. USA 79:6777-6781; Southern et al. (1982) J. Mol. APP. Genet. 1:327-341; PCAT Publication No. WO87/02062.
  • viral promoters e.g., SV40 early region promoter, Rous sarcoma virus, LTR promoter, etc.
  • Preferred eucaryotic expression vectors are those 5 employing the vaccinia virus, the SV40 virus, or the Rous sarcoma virus, which are also well known in the art. See, e.g., Mackett et al. (1984) J. Virol. 19:857; DNA Cloning: A Practical Approach, vol. II, pp. 191-211, (D. Glover, Ed.); PCT Publication No. WO86/07593; Chakrabaraty et al. (1985) Mol. Cell. Biol. 5:3403.
  • a shuttle vector will be employed where the vector is capable of replication in 5 different hosts requiring different replication systems. This may or may not require additional markers which are functional in the two hosts. Where such markers are required, these can be included in the vector.
  • the plasmid containing the vector, two replication systems and ° the marker(s) may be transferred from one host to another, as required.
  • any useful marker may be used. Desirably, resistance to neomycin or tetracycline are of interest.
  • a marker for selection is highly desirable for convenience, other procedures for 5 screening transformed cells have been described. See, for example, G. Reipin et al., Current Genetics (1982) 189- 193.
  • Transformed cells may also be screened by the specific products they make, for example, synthesis of a desired product may be determined by immunological or 0 enzymatic methods.
  • the desired DNA sequence may then be manipulated in a variety of ways to provide for expression of the Fc receptor. It is highly desirable that the three-dimensional structure of the Fc receptor gene expression product be retained, especially those regions responsible for binding to IgG. 0 Although there is no absolute method to check correct folding of the expressed protein, correct folding is generally verified by functional characterization.
  • the entire Fc receptor gene with its natural 5'- and 3'- regulatory regions may be introduced into an appropriate secretion expression vector.
  • further manipulation may be required.
  • the non-coding or 5' region upstream from the Fc receptor gene of interest may be removed by endonuclease restriction, Bal31 digestion, or the like.
  • the gene may be restricted and an adapter employed for linking the gene to the promoter region, where the adapter provides for the lost nucleotides of the Fc receptor gene.
  • the resulting plasmid will be cloned and isolated and, as required, the particular secretion vector component analyzed as to its sequence to ensure that the proper sequence has been obtained.
  • the desired sequence can be excised from the plasmid and introduced into a different vector, where the plasmid can be restricted and the secretion expression vector component manipulated, as appropriate.
  • the promoter or transcriptional regulatory region in procaryotic cells comprises nucleotide sequences which can effect the efficiency of transcription.
  • the sequences include regulatory regions at about -35 and -10 nucleotides from the start of the RNA chain.
  • the transcriptional regulatory region can also include regulatory sequences which allow the time of expression of the DNA sequence encoding the polypeptide of interest to be modulated by the temperature of the growth medium.
  • Illustrative transcriptional regulatory regions or promoters which provide for temperature-sensitive transcription include any promoters which will bind to the CI857 repressor, for example, the lambda left and right promoters, and the like. Synthetic promoters having sequences at least substantially similar to these sequences may also find use.
  • a preferred transcriptional regulatory region is the bacteriophage ⁇ P L promoter.
  • the transcriptional regulatory region additionally will include the bacteriophage 0 L operator.
  • the gene CI857 which codes for the temperature-sensitive CI repressor, will be included in the secretion expression vector. This will allow regulation of the promoter, by interaction between the repressor and the operator at low temperatures, for example, about 30°C. Increasing the temperature to about 42°C will inactivate the repressor and allow expression of the gene of interest.
  • regulatory sequences which allow the time of expression to be modulated include those which are modulated by growth medium components.
  • regulation of the lac or the trp-lac hybrid promoter may be accomplished by use of the gene for the lad repressor, which binds in the lac promoter region downstream from the -10 regulatory region.
  • the lad repressor gene may be present on an episome, preferably the laclq enhanced mutant, or can be included in the expression cassette itself. Provision of a significant concentration of the repressor in the growth medium inhibits the promoter function in the absence of inducers. Addition of inducers such as IPTG or lactose to the host cell growth medium enhances promoter function.
  • lactose may be used as an inducer instead of IPTG.
  • the transcriptional regulatory region may optionally include regulatory sequences which terminate transcription and which provide sequences or structures which inhibit degradation of the mRNA and thus increase the stability of the mRNA species and allow for higher expression.
  • procaryotic sequences are known, for example the Trp terminator, the gene 32(T4) terminator, or a synthetic terminator which is similar in sequence to gene 32.
  • Mammalian cells are a preferred eucaryotic expression host of the present invention.
  • Various cell lines and expression vectors are known in the art. Examples of appropriate mammalian expression hosts include kidney cell lines (e.g., human, murine or chicken embryo fibroblast cell lines) , Chinese hamster ovary (CHO) cells, HeLa cells, mouse NIH/3T3 and/or LMT K - cells.
  • kidney cell lines e.g., human, murine or chicken embryo fibroblast cell lines
  • CHO Chinese hamster ovary
  • HeLa cells e.g., mouse NIH/3T3 and/or LMT K - cells.
  • heterologous proteins can be expressed in myeloma cell lines employing immunoglobulin promoters. See, e.g., Banerjee, et al.. Cell (1983) 21:729-740; U.S. Patent No. 4,663,281.
  • Another preferred embodiment of the present invention is the expression of recombinant polypeptides in insect cells using viral vectors.
  • the viral vector baculovirus is used in such applications. For example, high levels of expression have been achieved with vectors based on Autographa californica nuclear polyhedrosis virus
  • Secretion of the Fc receptor can be achieved by using a signal sequence peptide.
  • the DNA sequence encoding the signal sequence is joined upstream from and in reading frame with a DNA sequence encoding the Fc receptor.
  • the signal sequence includes a cleavage site which is recognized by a signal sequence peptidase.
  • the DNA sequence encoding the signal sequence may be any DNA sequence obtainable from a gene that encodes a normally secreted polypeptide.
  • obtainable from a gene is intended a DNA sequence which can be obtained from a natural source, or a DNA sequence which is synthesized in whole or in part and has a sequence at least substantially similar to the DNA sequence obtained from a natural source.
  • the DNA sequence encoding the signal sequence may be identical to that of a naturally occurring sequence or may have changes in the sequence so long as the changes do not affect the ability of the resulting signal sequence to direct secretion of the polypeptide of interest.
  • signal sequences include the signal sequences from the ompA gene (Mowa, et al., Journal of Biochemistry (1980) 255:27-29); the phoA gene (Inouye, et al.. Journal of Bacteriology (1982) 149:434-439); the ompF gene (Itebgbeph, et al., Proc. Nat. Acad. Sci. fUSA) (1980) 77:2621-2625); the ompC gene (Mizuno, et al., FEBS Lett. (1983) 151:159-164; the phoE gene (Overbeck, et al., J. Mol. Biol.
  • signal sequences which may find use in the subject invention may be identified based upon the following characteristics.
  • the signal sequences are generally approximately of the same length, namely about 20 to 30 amino acids.
  • the amino acid sequence of a signal sequence starts with a segment of about 1 to 7 hydrophilic amino acids, the segment being terminated by a short amino acid sequence containing about 1 to 3 positively charged amino acid residues.
  • the remaining portion of a signal sequence is comprised of a hydrophobic region containing about 15 to 20 amino acid residues, commonly referred to as a hydrophobic core.
  • sequences may be identified by, for example, computer scanning to determine the hydrophobicity curve for an amino acid sequence encoded by an open reading frame.
  • the signal sequences will be prepared by synthesizing oligomers having the desired sequence as described above.
  • restriction sites may be included at the 5'r end and/or the 3'-end of the synthetic signal sequence to facilitate its insertion into an appropriate expression plasmid in conjunction with a DNA sequence encoding a polypeptide of interest.
  • Hot-sites are easily cleavable, either by chemical means or enzymatically.
  • Asn asparagine
  • Glycine glutamate
  • the protein can be cleaved by hydroxylamine under moderately mild conditions. To accomplish this, it must be assured that any internal sequence (Asn-Gly) of the protein of interest is removed by genetic modification.
  • all Asn in an Asn-Gly sequence was replaced with Gin.
  • a host cell which has been stably transformed by an expression cassette is selected to produce the recombinant polypeptide.
  • a stably transformed host is one wherein the expression cassette has integrated into the host cell's chromosome; however, in the case of bacteria or yeast expression hosts, it may be preferred to select expression hosts which maintain the cassette on a non-integrating episomal element, such as a plasmid.
  • the polypeptide is produced by growing host cells transformed by the expression cassette under conditions which cause the expression of biologically active polypeptide. The appropriate conditions to bring about expression are well known in the art, and depend primarily on the expression system and host selected. The polypeptide may be isolated from the host cells and purified.
  • the polypeptide can be purified directly from the growth media. If polypeptide is not secreted, however, it may be necessary to disrupt the host cells and purify the polypeptide from the cellular lysate. Various purification techniques, such as HPLC and immunoaffinity chromatography, are known, and the selection of the appropriate purification and recovery method is within the skill of the art.
  • Fc receptor gene has been introduced into an appropriate host, the host may be grown to express the gene.
  • the host may be grown to express the gene.
  • procaryotic and eucaryotic hosts can be employed.
  • Host cells can comprise any gram- negative organisms that do not have a temperature sensitive mutation, such as E. coli , for example JM109, JM107, JM105, JM101, DH5 ⁇ and the like.
  • the host cell may be grown to high density in an appropriate nutrient medium, according to methods known to those of ordinary skill in the art.
  • temperature-sensitive regulatory sequences have been included in the expression vectors, at the time it is desired to obtain expression of the DNA sequence encoding the polypeptide of interest, the temperature of the nutrient medium can be changed.
  • the host cells may be grown at a permissive temperature, generally about 30°C.
  • a permissive temperature generally about 30°C.
  • transcription from the P L promoter is repressed and the host cells may grow unhampered by the demands of the synthesis of a the foreign gene product, which additionally may be toxic to the host organism.
  • the temperature may be increased to a non-permissive temperature, for example, about 42°C, at which temperature 5 the C j 857 repressor is rendered inactive, permitting transcription from the P L promoter.
  • the transcriptional regulatory regions are inducible with a metabolic inducer such as lactose in a lac+ strain
  • the appropriate inducer can be added to the growth medium.
  • host cells which can be used with this system include DH1, DH5, GM48, JM107, RF or HB101. Laclv can be provided as a vector.
  • the cells are harvested, lysed and the product 5 isolated and purified by extraction, precipitation, chromatography, electrophoresis, and the like.
  • the cells are harvested and the expression product is liberated by destruction of the 0 cell wall, for example by hypotonic shock, and the like.
  • the expression product is secreted into the medium, the nutrient medium may be collected and the product isolated by conventional means, for example, affinity chromatography. To produce an ⁇ active protein it may be 5 necessary to allow the protein to refold, as is appreciated by those of ordinary skill in the art.
  • the peptides of the invention are used for 0 detecting the presence of and quantity of IgG in a biological fluid sample, such as by contacting the test sample with the peptide of the invention and detecting the presence or absence of a specific binding complex of the IgG in the test sample and the peptide of the invention.
  • the peptides of the invention are also useful as immunogens in the production of antibodies.
  • the peptides of the invention are very useful in the study of structure-function relationship of bacterial Fc receptors, since the chimeric molecule (SBG) does not have domain redundancy.
  • Diseases which are therapeutically-treated in the method of the invention include leukemia and lymphoma, cancer and other immune diseases, such as an autoimmune disease.
  • the individual whose cells are treated is preferably a primate, especially a human. Accordingly, the invention provides a new method for therapeutic treatment of cells of a host individual in need of treatment by administering the peptide of the invention.
  • the peptide of the invention can be administered alone or as a formulation in which an effective amount of the peptide is used in combination with at least one pharmaceutically acceptable diluent, carrier or adjuvant in order to facilitate its administration to an individual to be treated.
  • the diluent, carrier or adjuvant with which the peptide is mixed or formulated can be an inert solid or liquid of synthetic or natural origin and can be organic or inorganic in nature. Any of the materials customarily used for formulating pharmaceutically active proteins can be used.
  • suitable diluents, carriers or adjuvants include phosphate buffer saline (PBS) , water, salts, stabilizers, sugars and the like, as is known in the art.
  • a pharmaceutical preparation of the peptide with or without addition of a diluent, carrier or adjuvant.
  • Administration is by any of the conventional means of administering proteins to individuals and is preferably by parenteral or intravenous administration, but oral, topical, intralesional, or other known modes of administration can be used.
  • the effective amount of the peptide is the amount sufficient to produce the desired effect. This dosage can vary and can be dependent on various factors, including the method and conditions of application, the disease being treated, the diluent, carrier or adjuvant used and the like. Proper consideration of these factors to provide the sufficient effective amount of the peptide for administration to produce the desired disease treatment is within the skill of those versed in the art.
  • Restriction enzymes were purchased from New England Biolabs (Beverly, Massachusetts) , GIBCO/BRL (Grand Island, New York) .
  • Bacteriophage T4 DNA ligase and polynucleotide kinase were obtained from New England Biolabs (Beverly, Massachusetts) .
  • Calf intestinal phosphatase was obtained from Boehringer-Mannheim (Indianapolis, Indiana) .
  • [ 35 S]-methionine, [ ⁇ - 35 S]dATP, [ 32 P]ATP and [ 1 4C]-methionine protein standards were purchased from Amersham (Arlington Heights, Illinois) .
  • Rainbow protein standards were obtained from Amersham (Arlington Heights, Illinois) .
  • Acrylamide and urea were purchased from ICN (Costa Mesa, California) .
  • Agarose was purchased from Bio-Rad (Hercules, California) and FMC (Rockland, Maine) .
  • Ten-twenty percent gradient SDS-PAGE was obtained from ISS (Hyde Park, Massachusetts) .
  • Nitrocellulose filters and Elutip columns were purchased from Schleicher and Schuell (Keene, New Hampshire) .
  • a DNA sequencing kit was purchased from US Biochemicals (Cleveland, Ohio) .
  • X-ray film, CsCl, and phenol were purchased from Kodak (Rochester, New York) .
  • NenSorb columns were purchased from NEN Products (Boston, Massachusetts) .
  • E. coli K12, JM109, and JM107 were the host strains for the procedures.
  • the plasmids pUC18 and pUC19 were purchased from BRL (Grand Island, New York) and pKK223-3 from Pharmacia (Piscataway, New Jersey) .
  • Oligonucleotides were purified by electrophoresis on 20% acrylamide, 7M urea gels in 0.5 x TBE (50mM Triz a, 50mM boric acid, ImM EDTA) as described by Maniatis et al., A Laboratory Manual (Cold Spring Harbor Laboratory, 1982) . Bands migrating at the expected size were sliced from the gel, and the DNA fragments were eluted from the gel slice in extraction buffer (lOOmM Tris HCL pH 8.0, 5mM EDTA) overnight at 37°C. The eluted DNA fragments were purified on a NenSorb column (NEN Products, Boston, Massachusetts) , lyophilized and stored at -20°C in distilled water.
  • NenSorb column NenSorb column
  • DNA sequencing was performed on alkaline- denatured plasmid DNA using the protocol supplied with the US Biochemical Sequencing Kit. Samples were electrophoresed on 8% polyacrylamide-urea gels using a BRL apparatus.
  • Protein Sequencing was performed according to a method described by Speicher, et al., in Techniques in Protein Chemistry, pp. 24-35 (Hugli, ed. , Academic Press, San Diego, 1989) using an Applied BioSystems 477-A protein sequencer and Applied BioSystems reagents (Applied BioSystems 477-A protein sequencer and Applied BioSystems reagents (Applied BioSystems 477-A protein sequencer and Applied BioSystems reagents (Applied
  • synthetic protein G consists of two IgG binding domains (C2 and C3) and a proline-rich hydrophilic region at its C-terminal end, referred to as the W region.
  • the W region is specifically included in this gene for the purpose of immobilization of recombinant proteins to a solid support.
  • the amino terminal end of the gene contains the factor Xa cleavable site, designed to allow specific cleavage of fusion proteins.
  • the naturally occurring Asn-Gly sequences of protein SG have been converted to Gln-Gly in the synthetic proteins to remove internal hydroxylamine cleavage sites.
  • a NH 2 OH cleavable linker can be added to the N-terminal end of the gene to obtain specific cleavage.
  • Synthetic protein G (derived from the MD1 gene) and chimeric BG (SBG) (derived from the MD2 gene) were constructed from "cassettes" each encoding a different domain.
  • the MD-1 gene was constructed from three cassette segments SG-1, SG-2 and SG-3; the MD-2 gene was constructed from SB, SG-2 and SG-3 (see Figure 1) .
  • Each cassette in turn was assembled from different oligonucleotides as follows: SG-1, SG-2 and SB were each constructed from ten overlapping oligonucleotides, and SG-3, from six oligonucleotides ( Figure 2A, B, C & D) .
  • SG-1, SG-2, SG-3 and SB were prepared by 5' phosphorylation of each oligonucleotide and subsequent ligation with T4 ligase. Phosphorylations were done in a 0.01 ml volume containing 70 mM Tris-HCl, pH 7.6, 10 mM MgCl 2 , ImM ATP, 1 mM dithiothreitol, 25 units of T4 polynucleotide kinase (Pharmacia, Piscataway, New Jersey) and 200 p moles of each of the appropriate oligonucleotides were mixed.
  • pMDl and pMD2 are under the control of the tac (trp-lac) promoter.
  • E. coli cells containing these plasmids were grown in LB media (Miller, Experiments in Molecular Genetics (1972, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)) in the presence of 100 ⁇ g/ml ampicillin at 37°C.
  • LB media Miller, Experiments in Molecular Genetics (1972, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  • sample buffer 50 mM Tris HCl pH 6.8/20% (w/v) glycerol/2%(v/v) 2-mercaptoethanol/ 0.01% bromophenol blue
  • sample buffer 50 mM Tris HCl pH 6.8/20% (w/v) glycerol/2%(v/v) 2-mercaptoethanol/ 0.01% bromophenol blue
  • Aberrant molecular weights of truncated protein G have been reported previously (Guss, et al., Embo. J. (1986) 5:1567-1575).
  • a related proline-rich protein from Streptococcus pyrogens also has an apparent size on standard SDS-PAGE which is 17% larger than the size as deduced from the nucleotide sequence (Hollinghead, et al., J. Biol. Chem. (1986) 261:1677-1686).
  • the proline residues and the hydrophilic region may influence the migration rate in SDS-PAGE.
  • E. coli expressed proteins SG and SBG were purified by affinity chromatography on IgG Sepharose. IgG bound proteins were further purified by FPLC G-75 size exclusion. Details of the purification procedure are provided below. Purified proteins SG and SBG were used for the biological activity measurements.
  • a one liter culture of JM107 cells containing plasmids pMDl or pMD2 was grown to an optical density at 595 nm of approximately 0.7. The cells were then induced for three hours with IPTG at a concentration of 1 mM. The solution was pelleted, washed, and resuspended. The cell solution was French pressed (SLM Aminco, Urbana, Illinois) , and the supernatant fraction was passed through an IgG Sepharose 6 FF (Pharmacia,
  • Binding was also compared with native proteins A and G.
  • the amount of IgG required to inhibit binding of a conjugated rabbit IgG to different receptors was used as a standard, since it is known that protein A and protein G bind to rabbit IgG with equal affinity (Guss, et al., supra) .
  • the microliter plate was coated overnight with a receptor in PBS. Plates were washed (10 mM Tris, 150 mM NaCl, 0.1% Tween 20, 0.05% Thimerosal) and blocked with BLOTTO (0.5% dry milk in lOmM Tris, 150 mM NaCl, 0.1% Tween 20, and 0.05% Thimisol) .
  • the receptors were then bound to peroxidase-conjugated rabbit IgG alone, or in competition with other immunoglobulins.
  • the relative amount of competitor IgG required to give 50% inhibition of binding to the enzyme-conjugated rabbit Ig is reported in Fig. 8.
  • Polyclonal rabbit IgG was used, as it has been shown to bind to protein A and protein G with similar affinities. (Reis, et al., J. Immunol. (1984) 132:3098) The values given have been normalized to make the amount of unconjugated rabbit IgG giving 50% inhibition equal to one.
  • Results presented in Fig. 8 demonstrate that SG binds strongly to human IgG3, in contrast to recombinant wild-type protein A (rPA) and approximately equal to recombinant wild-type protein G (rPG) , while SBG shows intermediate binding. Similarly, both SG and rPG bind to IgG4 with lower affinity than rpA; again SBG shows intermediate binding. Therefore, as shown in the data presented in Fig. 8, synthetic protein G (SG) retains the IgG binding activity of the parental protein G, and synthetic BG (SBG) is structurally and functionally chimeric.
  • C2D2 peptide which includes the entire C2 repeat domain (55 amino acids) and a part of D2 unit (6 amino acids) in protein G to comprise a 61 residue-long molecule.
  • B fragment peptide the B homologous domain of protein A
  • Fc receptors SG and SBG have been constructed. These receptors contain several unique features; synthetic protein G receptor (SG) contains only two IgG binding domains from Streptococcal protein G. Chimeric receptor SBG contains one IgG binding domain of staphylococcal protein A and one binding domain of streptococcal protein G.
  • the Fc receptors of the subject invention can be loaded on the solid surface at higher density than protein A or G by virtue of their smaller size.
  • SBG is the smallest chimeric immunoglobulin-binding molecule that can be constructed.
  • These receptors are also unique in containing enzymatic cleavable linkers at their N- termini, as well as containing internal substitution of glutamine for asparagine in Asn-Gly sequence. While these modifications do not appear to affect the binding affinity, they make the proteins more versatile in their potential use for fusion construction, affinity purification, and site specific cleavage of the fusion proteins. Thus, these synthetic proteins provide novel Fc receptors and potentially useful plasmids for construction, expression and purification of fusion proteins.

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Abstract

L'obtention par synthèse chimique d'un gène codant pour les domaines de fixation de l'IgG de la protéine G et d'un gène codant pour un récepteur chimérique contenant un domaine de fixation de l'immunoglobuline G (IgG) commun aux protéines A et G. Ces gènes ont été assemblés à partir de trois cassettes codant chacune pour un domaine différent. La protéine synthétique G (SG) et la protéine chimérique BG (SBG) ont été insérées dans un vecteur plasmidique contenant un promoteur TAC (lymphocyte T activé) et un gène résistant à l'ampicilline, l'amplification et l'expression se faisant dans le E. coli. En plus des sites de fixation de l'IgG, les récepteurs Fc obtenus contiennent une terminaison de carboxyle hydrophile riche en proline assurant l'immobilisation sur un support solide, et un site clivable de facteur Xa permettant de les utiliser pour isoler et purifier par affinité d'autres gènes clonés à l'extrémité 5' de ces gènes. Ces gènes ont été exprimés dans le E. coli. L'activité biologique des protéines exprimées a été démontrée par immunoempreinte avec de l'IgG humaine ainsi que par test concurrent portant sur différents sous-groupes d'IgG humaine et recourant à un immunoadsorbant à fixation enzymatique.
PCT/US1994/009141 1993-08-23 1994-08-23 RECEPTEUR CHIMERIQUE CONTENANT UN DOMAINE DE FIXATION DE L'IgG COMMUN AUX PROTEINES A ET G WO1995006125A1 (fr)

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EP0752425A2 (fr) * 1995-06-21 1997-01-08 TECNOGEN Società Consortile per azioni Ligands peptidiques pour la région constante des immunoglobulines
JP2013521258A (ja) * 2010-03-05 2013-06-10 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング 抗体の選択的強化
JP2014100095A (ja) * 2012-11-20 2014-06-05 Kyushu Univ タンパク質検出用融合タンパク質およびタンパク質の検出方法
JP2014156428A (ja) * 2013-02-15 2014-08-28 Univ Of Tokyo 抗体結合タンパク質
CN105353129A (zh) * 2015-12-14 2016-02-24 北京华安麦科生物技术有限公司 一种蛋白g金黄色葡萄球菌肠毒素试剂盒及其制备方法
WO2016061427A1 (fr) * 2014-10-17 2016-04-21 The University Of Chicago Procédés et compositions impliquant des variants de la protéine g

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EP0550771A1 (fr) * 1991-07-25 1993-07-14 Oriental Yeast Co., Ltd. Proteine artificielle de combinaison avec les immunoglobulines
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ELIASSON M;ANDERSSON R;OLSSON A;WIGZELL H;UHLEN M;: "Differential IgG-binding characteristics of staphylococcal protein A, streptococcal protein G, and a chimeric protein AG.", JOURNAL OF IMMUNOLOGY., vol. 142, no. 2, 15 January 1989 (1989-01-15), BALTIMORE US, pages 571 - 581 *
ELIASSON M;OLSSON A;PALMCRANTZ E;WIBERG K;INGANAS M;GUSS B;LINDBERG M;UHLEN M ;: "Chimeric IgG-binding receptors engineered from staphylococcal protein A and streptococcal protein G.", JOURNAL OF BIOLOGICAL CHEMISTRY., vol. 263, no. 9, 25 March 1988 (1988-03-25), BALTIMORE US, pages 4323 - 4327 *
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0752425A2 (fr) * 1995-06-21 1997-01-08 TECNOGEN Società Consortile per azioni Ligands peptidiques pour la région constante des immunoglobulines
EP0752425A3 (fr) * 1995-06-21 1997-07-09 Tecnogen Scpa Ligands peptidiques pour la région constante des immunoglobulines
US6207807B1 (en) * 1995-06-21 2001-03-27 Tecnogen S.C.P.A, Method for the separation and purification of immunoglobulins
US6566077B1 (en) 1995-06-21 2003-05-20 Tecnogen S.C.P.A. Peptide useful as a ligand
JP2013521258A (ja) * 2010-03-05 2013-06-10 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング 抗体の選択的強化
US10894806B2 (en) 2010-03-05 2021-01-19 Boehringer Ingelheim International Gmbh Selective enrichment of antibodies
JP2014100095A (ja) * 2012-11-20 2014-06-05 Kyushu Univ タンパク質検出用融合タンパク質およびタンパク質の検出方法
JP2014156428A (ja) * 2013-02-15 2014-08-28 Univ Of Tokyo 抗体結合タンパク質
WO2016061427A1 (fr) * 2014-10-17 2016-04-21 The University Of Chicago Procédés et compositions impliquant des variants de la protéine g
US10759834B2 (en) 2014-10-17 2020-09-01 The University Of Chicago Methods and compositions involving protein G variants
CN105353129A (zh) * 2015-12-14 2016-02-24 北京华安麦科生物技术有限公司 一种蛋白g金黄色葡萄球菌肠毒素试剂盒及其制备方法

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