EP0560807A1 - Proteines de liaison d'immunoglobuline et molecules d'adn de recombinaison codant pour ces proteines - Google Patents

Proteines de liaison d'immunoglobuline et molecules d'adn de recombinaison codant pour ces proteines

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Publication number
EP0560807A1
EP0560807A1 EP19910920431 EP91920431A EP0560807A1 EP 0560807 A1 EP0560807 A1 EP 0560807A1 EP 19910920431 EP19910920431 EP 19910920431 EP 91920431 A EP91920431 A EP 91920431A EP 0560807 A1 EP0560807 A1 EP 0560807A1
Authority
EP
European Patent Office
Prior art keywords
glu
ala
asn
leu
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19910920431
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German (de)
English (en)
Inventor
Michael Graham University Of Southampton Gore
Andrew George Popplewell
Anthony Public Health Laboratory Atkinson
Christopher Roderick Public Health Goward
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Public Health Laboratory Service Board
University of Southampton
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Public Health Laboratory Service Board
University of Southampton
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Publication date
Priority claimed from GB909025666A external-priority patent/GB9025666D0/en
Application filed by Public Health Laboratory Service Board, University of Southampton filed Critical Public Health Laboratory Service Board
Publication of EP0560807A1 publication Critical patent/EP0560807A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/705—Fusion polypeptide containing domain for protein-protein interaction containing a protein-A fusion

Definitions

  • This invention relates to immunoglobulin-binding proteins and recombinant DNA molecules coding therefor.
  • Protein-A is a cell wall component of Staphylococcus auxeus which binds to the Fc region of inununoglobulins from a variety of sources (Langone, 1982). For example, it can bind to human IgG sub-classes 1, 2 and 4 but not in general to IgG 3 ,. It can also efficiently bind IgG from rabbit and pig, but it binds horse and cow IgG with lower affinity, and binds rat IgG only very weakly (Boyle and Reis, 1987).
  • SpA has also found use in chemotherapy to remove immune complexes from serum (see Palmer et al., 1989 and references therein), and in biotechnology where it has been incorporated into cloning vectors in which the cloned gene can be expressed as a fusion with SpA (Nilsson et al, 1985).
  • SpA B IgG binding domain-fragment B
  • the present invention has solved these problems by designing a synthetic Fc-binding domain which is highly amenable to site directed mutagenesis. Expression of polypeptides comprising this synthetic Fc-binding domain has enabled the production of immuno-globulin-binding proteins having distinct advantages compared to SpA, rendering them particularly useful in preparative and diagnostic techniques and in therapy.
  • the present invention provides a polypeptide capable of forming a complex with an
  • immunoglobulin said polypeptide being characterised by having at least 2, but not more than 4 binding domains, each capable of binding to the Fc region of an immunoglobulin of the IgG class.
  • polypeptide is characterised by having 2, but not more than 2 of said binding domains.
  • the binding domains possess a high degree of sequence homology with the binding domains of Staphylococcus aureus Protein-A (SpA).
  • SpA Staphylococcus aureus Protein-A
  • each of said binding domains has at least 75% sequence homology, preferably at least 90% sequence homology, with at least one of the binding domains designated A, B, C, D and E of Staphylococcus aureus Protein-A.
  • each of said binding domains has at least 75% sequence homology, preferably at least 90% sequence homology, with the binding domain designated B of Staphylococcus aureus Protein-A.
  • each of said binding domains consists of from 40 to 55 amino acid residues.
  • the following are especially preferred sequences for the binding domains of polypeptides according to the invention:
  • sequences consisting of at least 40 amino acid residues and derived from sequence (1) by
  • Polypeptides according to the invention having at least one binding domain as specified in alternative (2) above may be produced by site-directed mutagenesis, using as a starting point recombinant DNA molecules containing DNA sequences coding for sequence (1) above.
  • polypeptides according to the invention a binding capacity which has a different pH dependence compared to that of protein A itself. This may be achieved for example by replacing a non-ionisable amino acid residue in sequence (1) by an ionisable amino acid residue.
  • an ionisable residue may be replaced by a non-ionisable residue.
  • an ionisable residue may be replaced by another ionisable residue having a different pKa or pKb.
  • ionisable amino acid residues include
  • Tyr 18 (the Tyr residue which occurs in the partial sequence Ala Phe Tyr Glu) may be replaced by a residue selected from:
  • derived sequences (b) include sequences wherein the or at least one of the partial sequences
  • derived sequences (b) for the binding domains of polypeptides according to the invention are sequences having the following general sequence:
  • X can be phenylalanine, glutamic acid, histidine, cysteine or lysine.
  • Sequences for the binding domains of polypeptides according to the invention are preferably mutated by cassette mutagenesis.
  • each of the binding domains of the polypeptide of the invention has the same amino acid sequence.
  • each of said derived sequences is identical, i.e. the derived sequences contain the same amino acid substitutions) at the same position(s).
  • preferred polypeptides according to the invention having derived binding domain sequences as described above may exhibit modified, pH-dependent binding affinities.
  • Particularly preferred polypeptides according to the invention are provided at their C-terminal ends with an amino acid residue having a functional group allowing the polypeptide to be bound covalently to a solid support.
  • the polypeptides according to the invention are provided with a cysteine residue at the C-terminal end.
  • One such preferred polypeptide has the following C-terminal sequence
  • This polypeptide has two binding domains of formula (1) separated by a 7 amino acid linker (Lys Lys Leu Asn Glu Ser Gln) based upon the sequence linking adjacent IgG binding domains in native SpA.
  • the above polypeptide is further provided with a Cysteine residue at the C-terminus.
  • Preferred polypeptides according to the invention are produced in the form of fusion proteins, especially fusion proteins having a molecular weight in the range 18 - 30 kDa. It is further preferred that the fusion proteins of the invention comprise a polypeptide according to the invention, fused to an amino acid sequence capable of acting as a nucleus for protein folding events.
  • An example of such a sequence is the first 50 to 85 amino acids of DNase1.
  • said fusion protein is one having an N-terminal amino acid sequence comprising the sequence of the first 50 to 85 amino acids of DNase1, preferably the sequence of the first 8l amino acids of DNase1.
  • Said fusion proteins in accordance with the invention may further be in the form of inclusion bodies.
  • Preferred recombinant DNA molecules according to the invention are characterised by the presence of at least three, preferably four unique restriction sites, particularly restriction sites selected from DdeI MluI, BglII and MaeIII.
  • One such preferred DNA insert has the sequence
  • one or more codon may be replaced by a degenerative codon (i.e. one coding for the same amino acid).
  • the initial codon get in the first sequence (and the corresponding second codon in the second sequence) may be replaced by gcg, which is the codon present in the corresponding position in the sequence coding for the natural binding domain of SpA B .
  • Fc-binding proteins according to the invention designated 81-SpA B *-2 and 53-SpA B *-2 consisting of two such synthetic domains fused to part of the bovine DNAase1 gene
  • Figure 1 shows the complete nucleotide sequence and the encoded amino acid sequence of the synthetic SpA B * gene.
  • Figure 2 shows the formation of a gene encoding two SpA B * domains.
  • Figure 3a shows the construction of gene fusion plasmid p81-SpA B *-2.
  • Figure 3b shows the construction of gene fusion plasmids p81-SpA B *-2 and p53-SpA B *-2
  • Figure 4 shows the complete DNA and amino acid sequence of fusion protein 81-SpA B *-2.
  • Figure 5 shows an SDS-PAGE gel illustrating the time course of induction of fusion protein 81-SpA B *-2.
  • Figure 6 shows an SDS-PAGE gel illustrating inclusion body purification.
  • Figure 7 shows an enzyme linked immunosorbant assay for IgG binding.
  • Figure 8 shows the formation of 81-SpA B *-1 or
  • Figure 10 shows the affinities of 81-SpA B -2 and SpA for IgG at different pHs.
  • Figure 11 shows the relationship between binding of IgG by mutated 81-SpA B *-2 proteins and pH.
  • IgG-binding proteins (81-SpA B *-2 and 53-SpA B *-2) by total gene synthesis is described. Unique restriction sites have been placed along the genes to facilitate the production of variant proteins.
  • 81-SpA B *-2 is the product of the fusion of part of the gene for bovine DNAasel and a gene coding for the two B domains (SpA B ) of Protein A from bovine DNAasel and a gene coding for the two B domains (SpA B ) of Protein A from
  • the fusion product is expressed in high yields in Escherichia coli JM103 as an inclusion body which can be purified by centrifugation and washing with aqueous denaturants such as Triton and urea.
  • the protein may be extracted into 2.5M urea and IgG-binding activity is restored on removal of the urea by dialysis.
  • the protein has a single cysteine residue placed at the carboxyl terminal of the protein which facilitates either
  • the protein can be precipitated from solution by adjusting the pH to 6.0 and is very heat stable and loses no activity by heating at 85°C for 30 min. Variations of 81-SpA B *-2 have been produced by amino acid substitutions, and some of these mutated proteins show changes in IgG binding activity.
  • SpA B * was based upon one of the five IgG binding domains of Protein A; domain B (SpA B ) which has an amino acid sequence closest to the consensus sequence of the five domains. Further it is strongest binding of all isolated single domains.
  • the amino acid numbering system used to refer to residues in the synthetic binding domains throughout this description is based upon that devised by Uhlen et al (1984) and is shown in Figure 1 for ease of reference.
  • E. coli JM103 (Messing et al, 198l) was used as a bacterial host. Plasmid and phage vectors used were pUC19 (Yanisch-Perron et al, 1985) pkk223-3 (Brosius and Holy, 1984) and phage M13mp19
  • L-broth 1% bactotryptone, 0-5% yeast extract, 0.5% NaCl supplemented where appropriate with 50 ⁇ g/ml ampicillin (Sigma).
  • Restriction enzymes purchased from Boehringer Mannheim, Northumbria Biologicals Ltd
  • T4 DNA ligase T4 polynucleotide kinase
  • calf intestinal alkaline phosphatase Boehringer Mannheim
  • DNA sequencing was performed using 'Sequenase', modified phage T7 DNA polymerase (Tabor and Richardson, 1987; 'Sequenase' kit purchased from United States Biochemical Corporation). All sequencing protocols including template preparation, were performed according to the supplier's recommendations.
  • Oligonucleotides were synthesized on a fully automated Applied Biosynthesis 380A DNA synthesiser which employs the phosphoramidite method of solid phase synthesis (Atkinson and Smith, 1984). De-protected oligonucleotides were purified by electrophoresis on a 7M Urea 12% polyacrylamide gel from which the band corresponding to the full length DNA sequence was excised and eluted (Maniatis et al, 1983).
  • SpA B * A synthetic gene, termed SpA B * was constructed, based on the B domain of SpA.
  • the DNA sequence was modified to maximise where possible the codon usage for translation in E. coli (Guoy and Gautier, 1982;
  • Oligonucleotide cassette based site directed mutagenesis is facilitated by the introduction of a series of unique restriction sites at intervals in the DNA sequence.
  • SpA B * was constructed as a series of six oligonucleotides of length 58-66 bp (see Fig 1); adjacent
  • oligonucleotide pairs had a 7 bp cohesive overlap with the
  • the gly --> ala replacement occurs at a non-essential position in the 2nd ⁇ -helix (i.e. away from the face that interacts with Fc) and is not believed to affect Fc binding (Nilssen et al, 1987).
  • This substitution was done to remove the single Asn-Gly peptide bond, making the domain resistant to hydroxylamine treatment. This will permit the inclusion of such a bond at the junction between the DNAasel and SpA B moieties of the fusion protein so that the two may be split by hydroxylamine and separately purified.
  • the additional Cys is introduced at the C-terminus which is away from the Fc-binding region and provides a reactive site for possible fluorescent labelling or immobilisation onto Sepharose to give an IgG purification column.
  • a gene encoding two Fc-binding domains (SpA B *-SpA B *) was constructed by linking 2 of the SpA B * genes together by the
  • the synthetic linker DNA encodes those amino acids which separate adjacent Fc-binding domains in native Protein A. This technique also ensures that the cysteine residue and stop codons are removed from domain 1, giving an in-frame protein with a single C-terminal cysteine residue. This construction was also cloned into pUC19 to give plasmid pSpA B *-2.
  • SpA B * was designed with its own Shine-Dalgarno ribosome binding site ( Figure 1), high level expression was not achieved following sub-cloning into expression vector pkk223-3, so a gene fusion approach was used to increase expression.
  • the SpA B *-SpA B * sequence was linked to synthetically constructed genes encoding mutated and inactive bovine DNAase1 protein (Worrall and Connolly, 1990) to create two genes, the first encoding the first 81 amino acids of DNAase1 followed by a 12 amino acid spacer and then the 111 amino acids of SpA B *-SpA B * and the second
  • Gene fusion plasmids were constructed by purifying fragments of the SpA B *-2 gene from pSpA B *-2 and inserting these into
  • Figure 3b summarises the construction of the plasmids encoding two IgG binding domains and 81 or 53 residues from the N-terminus of the DNase1.
  • p81-SpA B *-2 was created by ligation of the Kpn I-Pst I fragment of pSpA B *-2 into Kpn I-Pst I cut pAW2.
  • p53-SpA B *-2 was constructed in the same way, using the XbaI-PstI restriction sites.
  • the plasmids encoding fusion proteins with single SpA B * domains were constructed by digesting each respective plasmid with Bgl II, removing the released fragment and religating the shortened, linearised plasmid. Restriction analysis and DNA sequencing were performed to confirm the generation of recombinant DNA molecules. The complete
  • nucleotide/amino acid sequence of the encoded fusion protein 81-SpA B *-2 is shown in Figure 4 and its amino acid composition is shown in Table 1.
  • 81-SpA B *-2 was produced as an inclusion body within the cell, a theory which was confirmed by microscopy (data not shown). When whole cells of an induced culture are disrupted by sonieation and then subjected to low speed centrifugation, 81-SpA B *-2 is found
  • Inclusion bodies were pelleted by centrifugation at 7.500 g for 15 minutes and were resuspended in 10 mM Tris-HCL pH 8.5, 1% v/v Triton X-100 detergent. The sonieation process was repeated to ensure that all cells had been disrupted, the sonicate was stirred at 4°C for 15 minutes then the inclusion bodies were pelleted by centrifugation as before. Two washes were performed in this way, then the pellet washed x 2 in 10 mM Tris-HCL pH 8.5 containing 1 M urea.
  • Solubilisation of the fusion protein was achieved by extraction in 10 mM Tris-HCL pH 8.5 containing 2.5 M urea at 4°C for 1 hour. The solution was spun at 27,000 g for 20 minutes and the supernatant was retained. A further extraction in 10 mM Tris-HCL pH 8.5 containing 4 M urea was performed on the pellets and again the supernatant was retained. The urea was dialysed away against 2 ⁇ 4 litres 20 mM KP buffer pH 8.0, and aliquots of the resulting protein solution were freeze dried.
  • the amino terminal 80-85 amino acid residues appear to exist as a domain distinct from the remainder of the protein. This domain includes several secondary structural features. Two a-helices (I and II, see Suck et al, 1984) consisting of residues 18-29 and 42-54 respectively are present, and six B-strands (A,B,C,D,E and F) four of which (A,C,E and F) form a B-pleated sheet and the other two form a parallel B-pleated sheet with each other.
  • N-terminal 81 residues of 81-SpA B *-2 or 2 may fold into the same. stable tertiary structure as in DNase 1.
  • the first 53 residues of DNase 1 (used in 53-SpA B *-1 or 2) also contain helices I and II but contain only four B-strands (A,B,C and D).
  • This truncated sequence may still fold into its 'native' structure but will be less stable having lost possible hydrogen bonds between strands C and F.
  • the existence of the complete and thus presumably more stable N-terminal domain of DNase 1 in 81-SpA B *-1 or 2 may act as a nucleus for protein folding events and hence maylead to a compact fusion protein, better protected from proteolysis.
  • An alternative method for protein concentration estimation is the bicinchoninic acid protein assay of Smith et al (1985) (Sigma).
  • IgG from other species was also shown to be bound by 81-SpA B *-2 by using competitive ELISA techniques.
  • competitive ELISA all wells were coated with 200ng of 81-SpA B *-2 in sodium carbonate buffer pH 9.6 as above. Serial dilutions of the test antibody in PBS-Tween were then made and allowed to bind to the 81-SpA B *-2 coating the wells for about 10min. 50 ⁇ l of Swine
  • the affinity for the IgG decreases in the following order:
  • conjugated swine-anti-sheep IgG from different species.
  • Experiment 2 used an identical procedure to Experiment 1, except that the serial dilutions of the test antibody in PBS-Tween were allowed to bind to the 81-SpA B *-2 coating the wells of the microtitre plate for 15 minutes, instead of about 10 minutes.
  • the results of this experiment are shown in Table 3 .
  • Table 3 Table 3
  • 81-SpA B *-2 human fc is added to 81-SpA B *-1 or any immunoglobulin or Fc is added to a solution of DNAasel.
  • both IgG binding sites in 81-SpA B *-2 are functional in the presence of human Fc or IgG but that there is either no affinity for IgG from chicken or that the complexes formed remain soluble.
  • Evidence from competitive ELISA experiments described above suggest that the former is the case.
  • the lack of formation of any precipitate with DNase 1 and IgG removes the possibility that the DNase 1 part of the fusion is involved in any binding phenomenon to either Fab or Fc.
  • the solution was agitated at intervals for 20-30 min, then the solution was discarded and the filter was washed 5 times in 0.1% v/v Tween 20 (in phosphate buffered saline (PBS) -Tween) and once in PBS.
  • PBS phosphate buffered saline
  • Chloronaphthol solution was added to cover the filter along with H 2 O 2 (to 0.1% v/v) and the solution was agitated. A purple/blue colour is indicative of IgG-binding activity.
  • precipitated 81-SpA B *-2 can be refolded to produce active protein by dissolving the precipitate in 4M urea and removing the urea by dialysis.
  • Mutants were produced by site-directed mutagenesis in which the amino acid residues designated 17 Phe and 18 Tyr (see below) were replaced as follows:
  • Figure 9 is a helical wheel representation of the amino acid residues in the SpA B domain shown by X-ray crystallographic studies of Melnhofer (1981) to fall into two helical secondary structural motifs.
  • Residue 18 in the helix closest to the amino terminus of the protein has been implicated to be essential for binding to the Fc of IgG and possibly to participate in a hydrogen bond (ring hydroxyl group) with the carbonyl group of the peptide bond formed between residue 432 and 433 of the IgG.
  • Figure 7 shows the level of porcine IgG-HRP activity retained per well containing various amounts of each IgG binding protein.
  • the data demonstrate clearly that the residue Tyr 111 (and 169) may be replaced by both Phe 111 (and 169) or Trp 111 (and 169) without severe disruption of the binding interactions.
  • Analysis of such binding data shows that replacement of Y111, Y169 with Fill, F169 causes a three fold decrease in affinity for the IgG, this being equivalent to a loss of 0.6kcal per mol of binding energy due to the loss of the intermolecular hydrogen bond per domain.
  • mutants with Tyr 18 replaced by other amino acids were made by digestion of the plasmid 81-SpA B *-2 with MlaI and Bgl II to release a short fragment encoding residues
  • the data displayed in Fig. 11 shows the amount of porcine IgG-HRP conjugate bound to various amounts of fusion proteins in the wells of a standard microtitre plate.
  • the mutant Y111H,Y169K shows less binding, approximately 8% of that of the native protein and the mutant Y111E.Y169E shows virtually no IgG binding under these conditions.
  • the interaction of all three proteins with Porcine IgG-HRP was found to be very sensitive to pH unlike the native protein. At pH values where the replaced residue would be expected to have a charge, the binding is less strong than under conditions where the equilibrium between charged and uncharged species lies towards the uncharged side.
  • the mutant Y111E,Y169E therefore shows maximal binding at pH 4 and minimal binding at pH 6 or above i.e.
  • both the mutants Y111H,Y169H and Y111K,Y169K show the increased binding of IgG-HRP at higher pH where both side chains become less protonated.
  • the apparent pK (6.5) of the binding curve shown by Y111H,Y169H is lower than that shown by
  • Table 4 gives the percentage of IgG binding shown by each mutant compared with the non-mutated construct at two pH values where the binding is minimal or maximum. It can be seen that the Glu mutant has a maximum binding of only 10% of the 'native' protein at pH 4.0 whereas much higher relative binding is obtained for the Lys (20%) or His (50%) mutants at pH 9.0.
  • the present invention has successfully overcome the problems of the prior art.
  • Recombinant DNA techniques are provided for the production of polypeptides having between 2 and 4 modified IgG binding domains which allow high levels of expression in E coli without incurring proteolysis by host enzymes or difficulties in purification.
  • Said binding domains are highly amenable to site-directed mutagenesis and therefore enable the production of immunoglobulin-binding proteins having distinct advantages compared to Protein A. Examples of such mutated proteins are given and their properties investigated.

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Abstract

Un domaine synthétique de liaison de F6 a été produit à partir d'au moins l'un des domaines de liaison de désignation A, B, C, D et E de la Protéine A de Staphylococcus aureus(SpA) au moyen de techniques de recombinaison d'ADN, et présente une réceptivité élevée à la mutagenèse dirigée.
EP19910920431 1990-11-26 1991-11-25 Proteines de liaison d'immunoglobuline et molecules d'adn de recombinaison codant pour ces proteines Withdrawn EP0560807A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB909025666A GB9025666D0 (en) 1990-11-26 1990-11-26 Production of immunoglobulin-binding protein by total gene synthesis
GB9025666 1990-11-26
GB9115814 1991-07-23
GB919115814A GB9115814D0 (en) 1990-11-26 1991-07-23 Immunoglobulin-binding proteins and recombinant dna molecules cloding therefor

Publications (1)

Publication Number Publication Date
EP0560807A1 true EP0560807A1 (fr) 1993-09-22

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EP (1) EP0560807A1 (fr)
AU (1) AU8957091A (fr)
CA (1) CA2096953A1 (fr)
WO (1) WO1992009633A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69230061T2 (de) * 1991-07-25 2000-01-05 Oriental Yeast Co. Ltd., Tokio/Tokyo Künstliches immunoglobulin-bindendes protein
JPH06263795A (ja) * 1992-09-30 1994-09-20 Kuraray Co Ltd ペプチドおよびこれを担体上に固定化してなる吸着剤
AU7563594A (en) * 1993-08-23 1995-03-21 Applied Immune Sciences, Inc. Chimeric receptor containing one igg binding domain of both protein a and protein g
SE9503925D0 (sv) * 1995-11-07 1995-11-07 Pharmacia Biotech Ab Separationsmedium för IgG
SE0301936D0 (sv) * 2003-06-30 2003-06-30 Affibody Ab New polypeptide
CA2791918C (fr) * 2010-03-05 2019-11-19 Boehringer Ingelheim International Gmbh Enrichissement selectif d'anticorps
LT3039033T (lt) * 2013-08-28 2019-10-25 Affibody Ab Surišantys polipeptidai, turintys mutuotą karkasą

Family Cites Families (6)

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Publication number Priority date Publication date Assignee Title
US5151350A (en) * 1982-10-27 1992-09-29 Repligen Corporation Cloned genes encoding recombinant protein a
US4691009A (en) * 1984-12-26 1987-09-01 Repligen Corporation Hybrid proteins produced by an ultrahigh prokaryotic expression system
US5089605A (en) * 1987-03-13 1992-02-18 Repligen Corporation Immobilized immunoglobulin-binding proteins
US5084559A (en) * 1987-03-27 1992-01-28 Repligen Corporation Protein a domain mutants
AU612370B2 (en) * 1987-05-21 1991-07-11 Micromet Ag Targeted multifunctional proteins
DE3887750T2 (de) * 1987-11-20 1994-09-15 Creative Biomolecules Inc Selektive entfernung von immunkomplexen.

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9209633A1 *

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CA2096953A1 (fr) 1992-05-27
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