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WO1992021972A1 - Methode de triage rapide utilisee dans l'identification d'interactions entre ligands et recepteurs - Google Patents

Methode de triage rapide utilisee dans l'identification d'interactions entre ligands et recepteurs Download PDF

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Publication number
WO1992021972A1
WO1992021972A1 PCT/US1992/004640 US9204640W WO9221972A1 WO 1992021972 A1 WO1992021972 A1 WO 1992021972A1 US 9204640 W US9204640 W US 9204640W WO 9221972 A1 WO9221972 A1 WO 9221972A1
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protein
receptor
fusion
binding
complex
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PCT/US1992/004640
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English (en)
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Roland Buelow
Julian K. Hickling
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Immulogic Pharmaceutical Corporation
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Publication of WO1992021972A1 publication Critical patent/WO1992021972A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • 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
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the field of this invention is the screening of diverse protein sequences.
  • polymorphic proteins In many physiological processes, there are involved a large number of polymorphic proteins. Illustrative of the diversity of biologically related compounds are components of the immune system, such as immunoglobulins, major histocompatibility complex (MHC) antigens, and T-cell receptors. This diversity is predicated on the need to be able to detect an extraordinarily large number of different compounds and proteins or other sequences, while still being able to discriminate between self and non-self.
  • MHC major histocompatibility complex
  • Methods are provided for screening large numbers of diverse poly(amino acid) sequences, where sequences undergo an initial enrichment, followed by screening of the diverse sequences with an appropriate receptor other than an immunoglobulin.
  • the enrichment is achieved by cloning the sequence as a fusion protein to a fusion partner.
  • the resulting clones are lysed and the fusion protein purified by complexation with a specific binding partner for the fusion partner.
  • the enriched protein fusion product may then be used directly for screening.
  • Figure 1 provides three graphs of the results obtained when mixing the lysates from pCA and pCA.HA and then subjecting them to various modes of purification.
  • the top graph (Fig. 1A) purifies the lysate mixture using a conventional antibody - Sepharose column; the middle graph (Fig. IB) uses Ig coated plate purification; and the bottom graph (Fig. 1C) uses BSA-anti BSA complex purification.
  • Methods are provided for screening large numbers of diverse protein sequences for binding to receptors, so as to evaluate ligand receptor interactions.
  • Open reading frames encoding diverse poly(amino acid) sequences are joined to a fusion partner to provide a sequence encoding a fusion protein.
  • the sequences are then cloned, so as to provide clones of a single fusion protein.
  • the cells from each clone may then be lysed and the lysate combined with a binding partner for the fusion partner, where the binding partner is polyvalent.
  • the resulting polyvalent complexes are separated from the lysate, washed and then used for screening of receptor binding, usually without further processing.
  • the sequences of interest may take many forms, being from diverse sources and may find use for diverse purposes. Sources of interest may include proteins or fragments thereof encoded by a cDNA library or genomic library, where the library may have been partially separated as to one or more properties.
  • the sequences may come from any cell
  • SUBSTITUTE type or organism where there is an interest in determining binding affinity for a receptor.
  • peptides and proteins may be prepared from oligodegenerate DNA sequences where the sequences are synthesized, and at one or more sites, two or more different nucleotides are added.
  • any mixture of nucleic acid sequences, naturally occurring, synthetic or combinations thereof, may be employed in the subject invention.
  • sequences of interest are joined to a sequence encoding a fusion partner.
  • the fusion partner will allow for binding of the amino acid sequence of interest to its receptor, expression of the fusion protein, formation of high molecular weight complexes in conjunction with a binding partner, and lack of cross-reactivity with the sequences of interest.
  • the fusion partners may come from diverse sources and provide for a variety of ways of forming a complex.
  • the binding partner will be polyvalent to allow for the formation of large complexes, when the fusion protein and the binding partner are brought together.
  • Various fusion partners may be employed, such as the functional portions or all of protein A, avidin, lectins, or any polyepitopic peptide or protein, or the like.
  • the binding partner may include sugars, where the sugars may be by themselves or in conjunction with larger aggregates, such as cells, polybiotin, where biotin may be bound to an oligomeric or polymeric backbone, or one or more antibodies, where the fusion partner is polyepitopic and has the same or different polyepitopic sites.
  • the open reading frame encoding the sequence of interest may be joined directly or through a bridge to the sequence encoding the fusion partner.
  • the bridge may or may not include a selective cleavage site, depending upon whether one wishes to isolate the peptide of interest separate from the fusion partner.
  • Various methodologies may be employed for providing for preferential, if not specific, cleavage between the peptide of interest and the fusion partner.
  • Enzymes of interest include Factor Xa, KEX2, HIV protease, and the like.
  • the gene encoding the fusion protein may be prepared in a variety of ways, employing synthesis using commercially available nucleic acid synthesizing equipment, where overlapping single stranded sequences can be prepared and then brought together and ligated. Alternatively, individual sequences may be cloned, undergo appropriate restriction and then ligated together. The polymerase chain reaction may also be used.
  • the particular manner in which the gene encoding the fusion protein is prepared " is not critical to this invention.
  • transcriptional initiation regions may be employed, which may be constitutive or inducible.
  • the promoters will be of weak or medium strength, where inducible promoters include promoters of ⁇ -galactosidase, cl ⁇ s and the like.
  • inducible promoters include promoters of ⁇ -galactosidase, cl ⁇ s and the like.
  • expression vectors normally have polylinkers downstream from a transcriptional initiation region, so that one may insert the coding region in appropriate juxtaposition to the functional sequences of the transcriptional initiation region.
  • Also present downstream will be a transcriptional termination region, which allows for processing of the transcribed sequence to a mature messenger RNA.
  • the vector will also usually include a marker for selection, preferably positive selection, which may include antibiotic resistance, complementarily to an auxotrophic host, or the like.
  • the marker will normally be under the control of a constitutive promoter.
  • Illustrative selective markers include amp, cam, neo, and the like.
  • At least one origin of replication which preferably allows for high copy number in the intended host.
  • either plasmid or phage replication systems may be employed, desirably plasmid replication systems.
  • the complete vector which may be used for cloning the various sequences will usually include a plasmid or viral origin, a transcriptional initiation region, and a sequence encoding the fusion partner under the regulation of the transcriptional initiation region. Downstream from the transcriptional initiation region may be a polylinker, either 5'- or 3'- to the fusion partner.
  • the polylinker will allow for insertion of sequences in reading frame with the fusion partner, frequently permitting random sequences to be inserted by allowing for insertion in all three reading frames to provide an open reading frame.
  • the sequence of interest will include an initiation codon or one may be provided as part of or upstream to the polylinker.
  • the subject vector may be introduced into the host by any convenient means, including transformation, electroporation, protoplast fusion, calcium precipitated DNA, or the like.
  • the particular host will be prokaryotic. While many different prokaryotes may find use, the host of choice will be E. coli. , particularly strains having the following mutations: recA. m ⁇ ' etc.
  • the cells may be cloned in accordance with conventional procedures, e.g., plating on agar, using limiting dilution, separation on gels, etc., growing the cells to high density, induction of expression of the fusion protein, as appropriate, harvesting the cells, and lysing the cells.
  • Various techniques for lysing may be used, although some techniques may be found less efficient than other techniques. Techniques which may be used include mechanical disruption, freezing and thawing, sonication, enzymes and/or detergent treatments, where the treatments may be used individually or in combination.
  • the lysates will then be combined with the binding partner of the fusion partner.
  • the particular type of complex is not critical to this invention and any convenient binding partner may be employed.
  • the binding partner may be used by itself or in conjunction with a second agent which provides for extensive cross-linking.
  • a second agent which provides for extensive cross-linking.
  • binding partners other than antibodies are employed, various techniques may be used to provide for extended complexation. With protein A, appropriate polyepitopic antigens may be employed, even including cells.
  • the immune complex may be preformed by combining the primary antibody with the secondary agent antibody to form a complex which may then be mixed with a lysate containing the fusion protein.
  • the large immune complexes or other large binding partner bind the fusion protein and may then be separated by any convenient means, conveniently centrifugation.
  • the complex particles which are isolated may then be washed, if desired, and re-suspended in medium, where the fusion protein is available for cellular interaction.
  • the lysate may be produced in situ by combining the cells with a cell wall degrading enzyme, such as lysozyme, following with detergent to allow for release of the fusion protein and binding to the binding partner bound to the surface.
  • a cell wall degrading enzyme such as lysozyme
  • the surface may then be washed and any non-specifically bound material removed to leave the surface free of contamination from components of the host.
  • the enriched fusion protein may now be used in a variety of ways. It may be screened directly with the receptor of interest. Alternatively, as already indicated, the peptide of interest may be cleaved from the fusion partner and complex, followed by isolation and purification, if desired. Various techniques exist for purification, such as gel electrophoresis, HPLC, and the like.
  • the enriched or purified fusion proteins or peptides of interest are then screened with the receptor, particularly the major histocompatibility complex Class I and II molecules and the T-cell receptor, more particularly T-cells or microsomes comprising the T-cell receptor.
  • the T-cells may be the T-cells from any particular source, such as peripheral blood, thymus, lymph node, pancreas, or other lymphoid tissue, may be homogenous or heterogenous, may be from the same or different species from which the sequences of interest were obtained, or the like.
  • APCs antigen presenting cells
  • T-cell response Various techniques exist for monitoring T-cell response.
  • thymidine uptake by employing radioactive thymidine
  • CD69 production or calcium flux.
  • a secretion product associated with T-cell activation or T-cell proliferation by measuring DNA replication, one can evaluate the presence of a sequence to which T-cells may respond. Since mixtures of clones may be employed, one can then select those samples in which a T-cell response has been observed and screen individual clones or smaller mixtures of clones, from one to ten clones, whereby one can identify the particular sequence which has provided the response.
  • a protein has been found to provide for a T-cell response
  • By providing for deletions in a nucleic acid sequence, which results in the deletion of several amino acids one can screen the entire protein by preparing clones of the mutated antigen and determining which clone(s) no longer has the ability to provide the T-cell response.
  • Deletions may include up to about 90% of the amino acids of a protein, with various strategies being employed to ensure identification of a sequence of interest.
  • a specific sequence of interest such as an immunodominant sequence which binds to a T-cell receptor
  • various receptors such as surface membrane proteins, as part of a cell membrane microsome or insoluble form, or immobilized soluble receptor, e.g., thyroxine binding globulin, or the like, to determine their biological activity.
  • a kit may be provided for convenience, comprising a vector comprising a prokaryotic replication system, e.g., origin of replication, a marker, an expression cassette comprising a transcriptional and translational initiation region—promoter and Shine-Dalgarno sequence—the sequence encoding the fusion partner, e.g. protein A, under the transcriptional control of said region, at least one restriction site, which allows for insertion in reading frame with the fusion partner, preferably a polylinker which allows for insertion in all three reading frames, a transcriptional termination region, and such other functional sequences as may be desirable.
  • the kit will also include a polyepitopic protein and antibodies to the protein, individually or as a complex, where one of the protein or antibodies may be bound to a surface.
  • the following examples are offered by way of illustration and not by way of limitation.
  • the pCA vector was prepared by employing the protein A encoding sequence from pRIT2T (Pharmacia) into pCDNA2 (Invitrogen) joined to a polylinker.
  • the plasmid had in the clockwise direction beginning with the protein A under the transcriptional control of the ⁇ -gal promoter region, a polylinker, the fl-origin, an ampicillin resistance gene, and a pBR322 origin.
  • the sequence coding for the hemagglutinin peptide 307-319 was inserted at BamHI and Xhol sites of the polylinker in reading frame with protein A to provide a fusion protein (pCA.HA) .
  • the plasmid pCA is transformed into E. coli. Tg-1 by electroporation.
  • the resulting transformants are grown overnight in 2xYT (tryptone-yeast extract containing 100 ⁇ g ampicillin/ml) and the resulting overnight culture used to inoculate 2xYT 1:50.
  • the second culture is grown until ODgoo ⁇ !•
  • IPTG isopropyl thiogalactoside
  • IPTG isopropyl thiogalactoside
  • the bacteria are lysed with lysozyme/detergent.
  • the lysate is cleared by centrifugation and stored at -20°C.
  • BSA-antibody complex purification of the fusion protein 3 ⁇ g of bovine serum albumin is incubated with 35 ⁇ g of affinity purified anti-BSA antibody in 55 ⁇ l phosphate buffered saline (PBS) for 2h at room temperature.
  • PBS phosphate buffered saline
  • the pCA lysate prepared above is thawed and spun for 30 min at 4°C.
  • To the immune complex prepared above is added 100 ⁇ l of the lysate and the mixture incubated for 30 min on ice. After spinning for 15 min at 4°C, the pellet is washed once with 500 ⁇ l sterile PBS and the pellet re-suspended in medium containing 5% human serum (for T-cell proliferation assays).
  • a 96 well flat bottom tissue culture plate is coated with 50 ⁇ l of 0.1 mg/ml human IgG in PBS overnight.
  • the wells are then washed 5X with PBS and coated with 50 ⁇ l of rabbit anti-human IgG in PBS (0.1 mg/ml), and the mixture incubated for lh at 4°C.
  • the wells are then washed 5X with PBS, and 150 ⁇ l of 25 mM Tris pH 8.0, 50 mM EDTA, 50 mM glucose and 5 mg/ml lysozyme added.
  • An overnight culture of pCA transformants is inoculated into 2xYT 1:10, the mixture allowed to grow for 2h at 37°C, expression induced by adding IPTG to 1 mM, and the culture allowed to grow for 4h.
  • 10 ⁇ l per well of the culture of peripheral blood lymphocytes is added to pre-coated wells, incubated 10 min at room temperature, followed by the addition of 20 ⁇ l of 10% Triton X-100, and the mixture incubated lh on ice. After washing 5X PBS, 100 ul antigen presenting cells (APC) and T-cells (1-2X10 4 ) (irradiated 0.5-1X10 5 PBL) is added per well.
  • One row of the plate is coated solely with antibodies as a control of the proliferation assay, while a second row is coated with pCA lysate.
  • the top graph in Figure 1 shows that in a mixture comprising 1000 parts of neat protein A to one part of protein A bound to amino acids 307-319 of hemagglutinin (HA307-319), one can detect the presence of the fusion protein at a concentration of 0.1 ⁇ l/well by using antibody complexes. Thus, at extraordinary low concentrations of the target fusion protein, one can still observe an effect in a T-cell response test using tritiated thymidine.
  • E. coli strain TGI (supE hsd ⁇ 5 thi ⁇ (lac ⁇ proAB) F' [traD36 proAB-i- lacl ⁇ J lacZ ⁇ M15] was used throughout (Immulogic Pharmaceutical Corp., Cambridge MA). T4 ligase, and all restriction enzymes were obtained from New England Biolabs (Beverley MA.). The construction of vector pCA, derived from pCDNA2 (Invitrogen, San Diego CA) , is described previously.
  • Oligonucleotides were synthesized using an Applied Biosystems (Foster City, CA) PCR-mate-EP 391 DNA synthesizer, and purified on OPC (oligonucleotide purification column) columns (Applied Biosystems) .
  • the oligonucleotides contained the sequence coding for the T cell determinant HA 307-319 of the influenza virus strain X31 hemagglutinin gene, followed by three stop codons (TAA, TAG TGA) , and a Bglll recognition sequence.
  • the 5' and 3' ends of the oligonucelotides were designed to be cohesive with BamHI and Xhol cut DNA respectively.
  • oligonucleotides were produced by inserting the codon NNK at the position to be mutated, where N represents an equal mixture of the oligonucleotides G,A,T and C, and K represents an equal mixture of G and C.
  • T cell clones and proliferation assays The T cell clone 18.41 specific for HA 307-319, isolated from a donor haplotype: HLA-DR4 Dw4, DR7, DRw53, DQw8, DQw9 was used for all the experiments in this study.
  • the clone recognized the peptide presented by DR4 Dw4, but not by DR1 Dwl, DR4 DwlO, DR4 Dwl4 nor DR4 Dwl5.
  • T cell clones were maintained in, and proliferation assays performed in RPMI1640 (Gibco, Gaithersburg MD) supplemented with 5% human AB serum (Biocell, Carson CA), 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 2mM L-glutamine (JRH Biosciences, Lenexa KS).
  • T cell proliferation assays were carried out in wells containing captured fusion peptide, by adding irradiated (3000 rads) DR4 Dw4 PBMC at 5 x 10 /well, followed by T cell clone 18.41 at 1 x 10 4 cell per well. The final well volume was 200 ⁇ l. The cells were incubated for a total of 72 hrs at 37°C, in a humidified, 95% air 5% CO2 atmosphere, and pulsed with tritiated thymdine (1 ⁇ Ci/well) (Amersham, Arlington Heights IL) for " the last 18 hrs of the incubation.
  • DNA was isolated from 1.5ml of an overnight culture to E. coli TGI by a modification of the STET boiling method (Golumbeski et al. (1990) U.S.B. Comments 16:4), and sequencing performed using Sequenase 2.0 (United States Biochemical Corp., Cleveland OH) .
  • oligonucleotides were synthesized as described in materials and methods, that allowed for substitution of every possible amino acid at position 319 within HA 307-319. 52 of the resulting colonies were selected at random, and the DNA sequenced. Nucleotide usage at position one and position three in the codon was random. At position two a deficiency in the frequency of cytidines incorporated was observed (% versus the expected 25%) . This bias was likely to have been introduced during the chemical synthesis of the oligonucleotide. Despite this, the observed amino acid usage at this position did not deviate significantly from that expected on the basis of random codon frequency (x 2 >> 0.05).
  • Position 319 is situated outside the core of the peptide shown to be responsible for binding to HLA molecules, and can be deleted without any loss of binding activity, providing the C terminus of the peptide is amidated. Thus, this position might be expected to be more tolerant of amino acid substitution than position 309. Indeed, the results showed this to be the case.
  • 114 stimulated proliferation greater than 75% of that obtained to the natural sequence, and only 22 colonies failed to stimulate clone 18.41 above background. Forty three colonies that stimulated proliferation above background were selected at random and sequenced.
  • Residues at position 310 and 316 of HA 307-319 are thought to interact both with the T cell receptor and the MHC molecule in the ternary complex, whilst position 313 is thought to interact solely with the T cell receptor.
  • Long chain biotin can be added to each of these positions without any loss of binding activity (Rothbard et al., supra) . and a variety of polar, non-polar, and charged substitutions can be made without affecting binding to DR4 Dw4.
  • the side chain requirements at each of these positions for T cell recognition are much more stringent.
  • Degenerate oligonucleotides were constructed which coded for amino acid substitutions at positions 310, 313, and 316 and screened as before. The results were graphed as previously described.
  • Position 313 is thought to interact solely with the T cell receptor.
  • Clone HA1.7 responded weakly (40-50% of response to natural sequence) to serine and phenylalanine monosubstitution, and failed to
  • Position 316 was more tolerant of amino acid substitution than 310 or 313, with more colonies stimulating proliferation above background levels. Seventeen out of 176 colonies gave 75% or more of the proliferation induced by the natural sequence, and these were found to code for amino acids; lysine (the natural residue), arginine, leucine, methionine and glutamine.
  • Position 316 is thought to pack against position 74 in the ⁇ chain of the Dr4 Dw4 molecule, and to be oriented up and over the HLA-DR ⁇ chain helix, imposing a requirement for an unbranched side chain (at least until the ⁇ carbon)
  • HLA-DR4 Dw4 position 74 is an alanine residue, explaining the requirement for a hydrophobic centre around the ⁇ and ⁇ carbon atoms at 316.
  • the fact that several side chain termini are tolerated suggests that the putative contact with the T cell receptor is not too rigorous, though acidic side chains clearly are not tolerated.

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Abstract

Méthode de triage rapide consistant à lyser des clones présentant une pluralité de séquences différentes renfermant potentiellement une séquence recherchée afin de stimuler les lymphocytes-T. Les séquences de poly(acides aminés) sont enrichies par clonnage des séquences jointes à la séquence codant la protéine A afin de produir une protéine de fusion pouvant être ensuite purifiée rapidement par liaison à des complexes immuns, et élimination des contaminants non spécifiquement liés. On peut utiliser la protéine fusionnée directement ou bien la séquence étudiée peut être clivée à partir de la séquence de la protéine A et ensuite utilisée.
PCT/US1992/004640 1991-06-06 1992-06-05 Methode de triage rapide utilisee dans l'identification d'interactions entre ligands et recepteurs WO1992021972A1 (fr)

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US10336808B2 (en) 2007-03-26 2019-07-02 Dako Denmark A/S MHC peptide complexes and uses thereof in infectious diseases
US10369204B2 (en) 2008-10-02 2019-08-06 Dako Denmark A/S Molecular vaccines for infectious disease
US10611818B2 (en) 2007-09-27 2020-04-07 Agilent Technologies, Inc. MHC multimers in tuberculosis diagnostics, vaccine and therapeutics
US10722562B2 (en) 2008-07-23 2020-07-28 Immudex Aps Combinatorial analysis and repair
US10968269B1 (en) 2008-02-28 2021-04-06 Agilent Technologies, Inc. MHC multimers in borrelia diagnostics and disease
US11992518B2 (en) 2008-10-02 2024-05-28 Agilent Technologies, Inc. Molecular vaccines for infectious disease
US12258373B2 (en) 2018-12-17 2025-03-25 Immudex Aps Panel comprising Borrelia MHC multimers

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US10336808B2 (en) 2007-03-26 2019-07-02 Dako Denmark A/S MHC peptide complexes and uses thereof in infectious diseases
US10030065B2 (en) 2007-07-03 2018-07-24 Dako Denmark A/S MHC multimers, methods for their generation, labeling and use
US10611818B2 (en) 2007-09-27 2020-04-07 Agilent Technologies, Inc. MHC multimers in tuberculosis diagnostics, vaccine and therapeutics
US10968269B1 (en) 2008-02-28 2021-04-06 Agilent Technologies, Inc. MHC multimers in borrelia diagnostics and disease
US10722562B2 (en) 2008-07-23 2020-07-28 Immudex Aps Combinatorial analysis and repair
US9404916B2 (en) 2008-09-20 2016-08-02 University College Cardiff Consultants Limited Use of a protein kinase inhibitor to detect immune cells, such as T cells
US10369204B2 (en) 2008-10-02 2019-08-06 Dako Denmark A/S Molecular vaccines for infectious disease
US11992518B2 (en) 2008-10-02 2024-05-28 Agilent Technologies, Inc. Molecular vaccines for infectious disease
US12258373B2 (en) 2018-12-17 2025-03-25 Immudex Aps Panel comprising Borrelia MHC multimers

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