WO2005035751A1 - Procede de selection rapide et efficace de proteines a haute fonctionnalite, proteines a haute fonctionnalite obtenues par ce procede, et procede de production et d'utilisation desdites proteines - Google Patents

Procede de selection rapide et efficace de proteines a haute fonctionnalite, proteines a haute fonctionnalite obtenues par ce procede, et procede de production et d'utilisation desdites proteines Download PDF

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WO2005035751A1
WO2005035751A1 PCT/JP2004/015290 JP2004015290W WO2005035751A1 WO 2005035751 A1 WO2005035751 A1 WO 2005035751A1 JP 2004015290 W JP2004015290 W JP 2004015290W WO 2005035751 A1 WO2005035751 A1 WO 2005035751A1
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protein
library
antibody
minutes
molecule
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PCT/JP2004/015290
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Japanese (ja)
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Hiroshi Yanagawa
Noriko Tabata
Kanehisa Kojoh
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Keio University
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Publication of WO2005035751A1 publication Critical patent/WO2005035751A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1062—Isolating an individual clone by screening libraries mRNA-Display, e.g. polypeptide and encoding template are connected covalently

Definitions

  • the present invention relates to a method for selecting a highly functional protein, a highly functional protein obtained thereby, and a method for producing and using the same.
  • Non-Patent Document 4 a method for single-chain antibodies (Non-Patent Document 4) in which the C-terminal of one chain is linked to the N-terminal of the other with a very flexible peptide linker! / ⁇ You. [0004]
  • these techniques still have some disadvantages.
  • the phage display uses a realistic DNA library with an upper limit of about 10 9 (for antibodies), small library size, and the use of biological systems (e.g., E. coli infection, amplification, etc.). Will have an unexpected bias (mutation, etc.).
  • coli cannot be used. Since ribosome display is a complete in vitro experimental system using a cell-free translation system, these biases are ineffective, and the upper limit of the library can be 10 1 Q -10 U. However, the formation efficiency of the mRNA-protein-ribosome ternary complex is extremely low at 0.2% (Non-patent document 3), and is several hundredths of that of the in vitro viru S (IVV) method (Non-patent document 5). It is. In addition, since the complex is very unstable, there is a problem that a stronger selection pressure cannot be applied during the biobanning operation.
  • the selection pressure in the banner is the most important factor in the antibody concentration, and if the selection pressure is not strongly applied, it becomes necessary to repeat more selection cycles.
  • the selection pressure since a wide variety of antibodies with different stability and affinity can be obtained for one type of antigen, it is necessary to clone, express, and analyze many samples in order to obtain more functional ones. And This is not only a lot of hard work, but also time and money.
  • Non-Patent Document 6 As cell-free translation systems for synthesizing proteins in vitro, large-scale expression of proteins has been studied in a wheat germ system (Non-Patent Document 6) and an E. coli system (Non-Patent Document 7). ing. Along with this, 3'UTR is generally used to improve the stability and translation efficiency of mRNA as a stable translation template capable of expressing a large amount of protein (Non-Patent Document 8). A method such as substitution or modification of the chemical structure of mRNA (Non-Patent Document 9) is used.
  • RNA-protein linked molecule (IW) in which protein and mRNA are covalently linked via puromycin by performing a cell-free translation reaction using type III mRNA containing a piuromycin bound to the 3 'end.
  • Library can be constructed. After screening in vitro proteins that bind to the target molecules of this library of mapping molecules, the genes can be amplified and decoded by reverse transcription PCR. After that, we performed a proteome analysis of the IW method.
  • Non-Patent Document 5 a type II DNA that can express IVV and C-terminally labeled proteins with high efficiency using a cell-free translation system of wheat germ extract.
  • the present inventors have previously described a method for efficiently constructing IW, an assigning molecule, by linking mRNA (genotype) and protein (phenotype) to a cell-free translation system via pure bitemycin.
  • a large amount of protein is required for detailed function and three-dimensional structure analysis of proteins and production of antibodies. For this reason, it is difficult to purify the endogenous target protein and obtain a required amount thereof. Therefore, a recombinant protein in which cDNA of the target protein is expressed in a suitable host and used is used.
  • Various hosts for expressing a single-chain antibody can be considered, such as Escherichia coli, yeast, insect cells, and animal-derived cultured cells. Among them, E. coli is convenient because a large amount of protein can be obtained in a short period of time at low cost.
  • the use of a system that infects insect cells with a recombinant baculovirus allows expression of proteins that is difficult with the E. coli system.
  • Antibodies play an important role in various analytical tools.
  • One of them is the detection and quantification of proteins.
  • Methods for detecting proteins using antibodies include in vitro methods such as Western blotting, immunostaining, fluorescent antibody staining, and antibody chip methods, and in vivo methods such as immunoprecipitation.
  • the antibody In order to detect proteins using such a technique, the antibody must be pre- An emitted protein, for example, GFP is fused, or an antibody is fused to an enzyme protein, for example, horseradish peroxidase or alkaline phosphatase, and the enzyme activity is used as an index.
  • Methods for detecting protein-protein interaction using antibodies include surface plasmon resonance, fluorescence resonance energy transfer, fluorescence depolarization, evanescent field imaging, fluorescence correlation spectroscopy, fluorescence imaging, and solid-phase imaging.
  • enzyme immunoassays and the like Fluorescence Correlation Spectroscopy (FCS) requires a small amount of sample (approximately femtoliter), requires a short measurement time (approximately 10 seconds), and is easy to automate for HTS.
  • FCS Fluorescence Correlation Spectroscopy
  • EVOTEC is developing a device aiming for Ultra HTS, which screens more than 100,000 samples per day), and is superior as a detection system (Non-Patent Document 13).
  • the IW method is a method for constructing an mRNA-protein linking molecule in which mRNA and a protein are chemically linked via puromycin on the ribosome when mRNA is expressed in a cell-free translation system or the like ( Non-patent document 10; Non-patent document 12; Non-patent document 5). Furthermore, the mRNA-protein-linked molecule can be obtained by in vitro selection, and the mRNA portion of the selected assigning molecule can be amplified by reverse transcription PCR.
  • DTT dithiothreitol
  • a protein having an SS bond represented by an antibody since various commercially available cell-free translation systems contain DTT (dithiothreitol) as a reducing agent, they are not suitable for expression of a protein having an SS bond represented by an antibody.
  • DTT is essential for the expression of the wheat germ cell-free translation system, screening and expression examples of antibodies with high binding activity from single-chain antibody cDNA libraries have been Absent. Therefore, it is desired to establish a wheat embryo cell-free translation system capable of screening for a desired antibody having a very high binding activity even in the presence of DTT.
  • Non-Patent Document 2 Smith, G.P. (1985) Science 228, 1315-1317
  • Non-Patent Document 3 Hanes, J. and Pluckthun, A. (1997) Proc. Natl. Acad. Sci. USA 94,
  • Non-Patent Document 4 Huston, J.S., Margolies, M.N., Haber, E. (1996) Adv. Protein Chem., 49, 329.
  • Non-patent document 5 Miyamoto-Sato, E., et al., (2003) Nucleic Acids Res., 31, e78
  • Non-patent document 6 Madin K, et al. (2000) Proc. Natl. Acad. Sci. USA ., 97, 559-564
  • Non-Patent Document 7 Shimizu, Y. et al. (2001) Nat.BiotechnoL, 19, 751-755.
  • Non-Patent Document 8 Sachs.A.B., Et al. (1997) Cell 89, 831-838
  • Non-Patent Document 9 Ueda. T "et al. (1991) Nucleic Acids Symp Ser. 25, 151-152
  • Non-Patent Document 10 Nemoto, N., et al., (1997) FEBS Lett., 414, 405- 408
  • Patent Document 1 JP-A-10-816636
  • Patent Document 2 International Publication No. W098 / 16636 pamphlet
  • Patent Document 3 Japanese Patent Application Laid-Open No. 2002-176987
  • Patent Document 4 International Publication No. WO02 / 48347 pamphlet
  • Non-patent literature l l Nemoto, N., et al. (1999) FEBS Lett. 462, 43-46
  • Non-patent Document 12 Miyamoto-Sato, E "et al. (2000) Nucleic Acids Res. 28, 1176-1182
  • Patent Document 5 JP-A-11-322781
  • Patent Document 6 JP-A-2000-139468
  • Patent Document 7 International Publication WO02 / 46395 pamphlet
  • Non-Patent Document 13 Masataka Kaneshiro (1999) Protein nucleic acid enzyme 44: 1431-1438
  • the present inventors believe that, when a library of nucleic acids encoding a protein is prepared by the IW method, the target is not obtained conventionally by inactivation of the protein in the selection of the protein. As a result, they have found that heat treatment at such a high temperature can apply a high selection pressure, and as a result, a high-functional protein can be selected quickly and efficiently. In addition, they have found that a high-functional protein can be selected even when a protein having an SS bond is translated by a cell-free translation system that requires a reducing agent. The present invention has been completed based on the above findings.
  • the present invention provides the following.
  • a method for selecting a protein that interacts with a target molecule or a nucleic acid encoding the same comprising the following steps (a) to (d).
  • a method for producing a protein that interacts with a target molecule comprising: (1) selecting a nucleic acid encoding a protein that interacts with the target molecule by any one of the selection methods (1) and (9); A production method comprising a step of producing a protein by translating a selected nucleic acid.
  • step of producing a single-chain antibody comprises translating the selected nucleic acid with a cell-free translation system containing a thiol compound.
  • the step of producing a single-chain antibody includes producing as a fusion protein a single-chain antibody encoded by the selected nucleic acid and an enzyme or green fluorescent protein (GFP). (11) Manufacturing method.
  • a single-chain antibody having angiotensin II-binding activity which has the amino acid sequence shown in the following (A) or (B):
  • SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 or Pamino 105 is an amino acid rooster.
  • a single-chain antibody having binding activity to Lewis X which is represented by the following (A) or (B): A single-chain antibody having an amino acid sequence.
  • Fluorescence correlation spectroscopy Fluorescence correlation spectroscopy, fluorescence imaging analysis, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, or enzyme-linked immunosorbent assay (25) Method.
  • a therapeutic agent comprising the antibody of (27) as an active ingredient.
  • FIG. 1 Construction of a single-chain antibody cDNA library used in the present invention.
  • FIG. 3 Chemical structural formula of angiotensin II-pyotin.
  • MI2 MI1 was reacted in [18] at 50 ° C for 30 minutes, then at 99 ° C for 5 minutes, and angiotensin II was selected as an antigen and collected in [25].
  • MI3 A library collected by reacting MI2 in [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, selecting angiotensin II as an antigen, and collecting in [25].
  • FIG. 6 The translation solution obtained in [15], in which the corresponding molecules were confirmed by 5% polyacrylamide electrophoresis in the presence of 8M urea (electrophoresis photograph).
  • MHO Library obtained in [11]; MK1:
  • the library obtained in [11] was reacted in [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, and Lewis X was selected as an antigen, and the library recovered in [25]; MK2: MK1 In [18], the library was reacted at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, and Lewis X was selected as an antigen, and the library recovered in [25].
  • FIG. 7 The translation solution obtained in [15], in which the corresponding molecules were confirmed by 5% polyacrylamide electrophoresis in the presence of 8M urea (electrophoresis photograph).
  • MM1 The library obtained in [11] was reacted at 50 ° C for 30 minutes in [18], and angiotensin II was selected as an antigen, and the library was recovered in [25];
  • MM2 MM1 was recovered in [18].
  • FIG. 8 The eluate obtained in [21] was subjected to PCR in [22] and subjected to 1% agarose gel electrophoresis. The top is an electrophoresis gel (electrophoresis photograph). The lower bar graph shows the absorption of ethidium bromide in the upper electrophoresis gel measured and quantified using MOLECULAR IMAGER FX (Bio-rad).
  • FIG. 11 A phylogenetic tree created by TreeViewPPC after sequence alignment by clustak based on amino acid sequences for in-frame results obtained by performing sequence analysis in [31].
  • a surrounded by a line indicates the one converged by the selection of Angiotensin II, and B surrounded by a line indicates the one converged by the selection of Lewis X.
  • the number of the clone was shown after the library abbreviation.
  • FIG. 12 is an enlarged view of A and B surrounded by the line in FIG. A: Converged by Angiotensin II selection, B: Converged by Lewis X selection.
  • FIG. 13 Western blot results (electrophoresis photograph). The right two are Western blots of MI3-55 [37]. Left three are Western blot controls.
  • FIG. 14 A bar graph showing the results obtained by translating the sequence obtained by selection using angiotensin II as an antigen in [34] and then analyzing it in Biacore in [39].
  • FIG. 15 A bar graph showing the result obtained by translating the sequence obtained by selection using Lewis X as an antigen with [34] and then analyzing with Biacore [39].
  • FIG. 16 A bar graph showing the results of translating MI3-55 at [34], treating it at 4 ° C. or 60 ° C. or 99 ° C. for 5 minutes, and analyzing with Biacore of [39].
  • FIG. 17 A bar graph showing the result of translating MI3-55 at [34], treating it at 4 ° C. or 60 ° C. or 99 ° C. for 5 minutes, and analyzing the result by ELISA [40].
  • the method for selecting a protein that interacts with a target molecule or a nucleic acid that encodes the protein according to the present invention is characterized in that the nucleic acid portion of the assigning molecule in the library encodes a protein that interacts with the target molecule; Except for subjecting the library of molecules to heat treatment, the method can be performed according to a conventional nucleic acid selection method using a corresponding molecule (hereinafter, also referred to as "IW method").
  • Target molecule means a molecule that interacts with a protein to be selected, and specifically includes proteins, nucleic acids, sugar chains, low molecular weight compounds, and the like.
  • selection vs. As long as it has the ability to interact with the elephant protein, there is no particular limitation on the full length of the protein or a partial peptide containing a binding active site.
  • the protein may be a protein whose amino acid sequence and function are known or unknown. These can be used as a target molecule even with a synthesized peptide chain, a protein purified from a living body, or a protein translated using an appropriate translation system such as a cDNA library, and purified.
  • the synthesized peptide chain may be a glycoprotein having a sugar chain bonded thereto.
  • a protein which is translated and purified using a suitable method for a purified protein having a known amino acid sequence or a cDNA library can be used.
  • the nucleic acid any DNA or RNA can be used as long as it has an ability to interact with the protein to be selected.
  • the nucleic acid may have a known nucleotide sequence or function, or may have an unknown nucleic acid.
  • those having a function as a nucleic acid capable of binding to a protein and having a known base sequence, or those that have been cut and isolated using a genomic library first-class restriction enzyme or the like can be used.
  • the sugar chain is not particularly limited as long as it has the ability to interact with the protein to be selected, and the sugar sequence or function may be a known sugar chain or an unknown sugar chain.
  • a sugar chain which has already been separated and analyzed and whose sugar sequence or function is known is used.
  • the low molecular weight compound is not particularly limited as long as it has the ability to interact with the protein to be selected. Either those whose functions are unknown or those whose ability to bind to a protein is already known can be used.
  • reaction between these target molecules and the target protein of the present invention or “interaction” between the target molecules usually means a covalent bond, a hydrophobic bond, a hydrogen bond, a Van der Waals between two molecules. At least one of a bond and a bond by electrostatic force. A force indicating an action by a force acting between molecules. This term should be interpreted in the broadest sense, and should not be interpreted restrictively in any sense.
  • the covalent bond includes a coordinate bond and a dipole bond.
  • the coupling by electrostatic force includes not only electrostatic coupling but also electric repulsion.
  • a binding reaction, a synthesis reaction, and a decomposition reaction resulting from the above-described actions are also included in the interaction.
  • Specific examples of the interaction include binding and dissociation between an antigen and an antibody, and a protein receptor. Binding and dissociation between a molecule and a ligand, binding and dissociation between an adhesion molecule and a partner molecule, binding and dissociation between an enzyme and a substrate, binding and dissociation between a nucleic acid and a protein bound thereto, proteins in a signal transduction system Binding and dissociation between glycoproteins and proteins, binding and dissociation between sugar chains and proteins, or binding and dissociation between low molecular weight compounds and proteins. Can be
  • the IW method is much more stable than ribosome display or phage display because the mRNA is covalently bound to the protein via the protein synthesis inhibitor puromycin or a derivative thereof. Therefore, it is possible to perform a higher selective pressure baning than the two techniques of ribosome display and phage display, thereby selecting not only specificity but also a protein that interacts with a more stable target molecule. In addition, the desired protein can be obtained in a smaller number of selection cycles because of the higher and higher concentration effects. Furthermore, it is not necessary to analyze many samples because it is concentrated to almost single. With these, the operation is simple, and the time and cost can be greatly reduced.
  • the establishment of highly functional antibodies (specificity, affinity, stability, etc.) in the library depends on the size (diversity) of the library.
  • the upper limit of the selectable library of the IW method can be 10 13 or more if conditions are satisfied, and a library larger than any other method can be targeted.
  • unnecessary bias is not applied.
  • the obtained protein can be expressed as a functional protein in a cell-free translation system containing a reducing agent such as DTT, it is possible to prepare a large number of samples, thereby facilitating high-throughput filtration.
  • the protein thus selected can be used in various fields depending on its function.
  • the high affinity single-chain antibody thus selected can be used for extracellular or intracellular protein detection and interaction detection.
  • the high-affinity single-chain antibody which has been selected for the mouse antibody cDNA library, is replaced with a human IgG antibody variable region and CDR region (complementarity determining region) to produce a chimeric IgG antibody or a humanized mouse antibody.
  • Make IgG antibodies These antibodies produce little or no human anti-mouse antibody when elicited when administered to humans .
  • a high-affinity single-chain antibody which has been selected as a human antibody cDNA library, can produce a fully human monoclonal IgG antibody by replacing the variable region of the human HgG antibody. This can be used as an antibody drug that does not cause anaphylactic symptoms.
  • the single-chain antibody used as the protein to be selected in the present invention has a V chain and a V chain
  • the DNA library encoding a single-chain antibody used in the present invention is not particularly limited as long as it is a library containing DNAs encoding a large number of single-chain antibodies. In particular, it is preferable to use one containing DNA encoding more than 10 9 different antibodies, which can correspond to any antigen.
  • Powerful antibody DNA libraries include, but are not limited to, those derived from higher vertebrates, preferably mouse, human, -petri, goat, camel spleen and B cells, used in conventional in vitro antibody selection systems, including commercially available ones. Any natural antibody DNA library may be used.
  • a mouse native antibody cDNA library (Krebber, A. et al. (1997)) J. Immun. Methods. 201, 35-55: Engberg, J. (1996) Molecular Biotech. 6, 287-310) MRNA was extracted from the spleen of the mouse, and the gene fragments encoding the variable regions (V, V) of the antibody H and L chains were analyzed by RT-PCR.
  • the obtained fragment force is also the C-terminal of V
  • the mouse single-chain antibody cDNA library is completed.
  • any linker sequence can be used as long as it has a relatively high degree of freedom, but it is generally preferable to use a (Gly Ser) sequence (SEQ ID NO: 120).
  • the order of joining is V
  • any mutant type antibody DNA library can be advantageously used.
  • any mutant type antibody DNA library can be advantageously used.
  • any mutant type antibody DNA library can be advantageously used.
  • n-CoDeR A library that extracts only CDRs (three H chains and three L chains) from antibody cDNA by PCR and incorporates them into an antibody frame that is easy to express in E. coli and phage (n-CoDeR: diverse Sex is 2 X 10 9 ) (Jirholt, P. et al. (1998) GENE 215, 471-476:
  • Step (a) is a step of preparing a library of DNAs encoding proteins that interact with the target molecule.
  • a library of DNAs encoding this protein can be prepared according to a conventional method.
  • step (b) the DNA of the library prepared in (a) is transcribed, and a spacer containing puromycin is ligated to the 3 'end of the transcribed RNA (hereinafter, prepared here). Is sometimes referred to as a “translation template”), and then a library of mapping molecules is prepared in a cell-free translation system by preparing mapping molecules for genotypes and phenotypes. You.
  • This step can be performed in the same manner as in the ordinary IW method.
  • the force spacer which will be described in more detail below, preferably contains polyethylene glycol.
  • the step (c) is a step of subjecting the library of assigning molecules to heat treatment.
  • the heat treatment means exposure to heating conditions that would not be imposed by the treatment in the normal IW method.
  • the assigning molecule is directly bound to the target molecule after translation, it is necessary to expose the subject to a heating condition that does not exist during translation, and the RNA portion of the assigning molecule is converted into an RNA-DNA hybrid by reverse transcription.
  • exposure is to heating conditions that are not between translation and reverse transcription.
  • This temperature is typically 50-100 ° C for 1-30 minutes, depending on the process of translation and force binding to the target molecule. Selected from a range.
  • heat treatment is preferably performed at a temperature of 80 ° C or higher, more preferably 90 ° C or higher, regardless of whether there is only translation or translation and reverse transcription.
  • the RNA portion of the assigning molecule be converted into an RNA-DNA hybrid by reverse transcription before the step of (U. This prevents nonspecific binding of the RNA to the carrier and the target molecule.
  • reverse transcription it is preferable to remove a substance derived from a cell-free translation system that inhibits the reverse transcription reaction before reverse transcription. , A spin column, a nickel column and the like.
  • the order of the heat treatment, the reverse transfer, and the fractionation is not particularly limited, but usually, the order of the heat treatment, the fractionation, and the reverse transfer, or the order of the fractionation, the reverse transfer, and the heat treatment is generally. preferable.
  • Step (d) is a step in which the assigning molecule is bound to the target molecule, washed sufficiently, then eluted, and the nucleic acid portion of the assigning molecule is amplified by reverse transcription-PCR or PCR. This step can be performed in the same manner as in the ordinary IW method. Elution can usually be performed by elution with a solution containing a free target molecule.
  • a process for using the DNA amplified in (d) as a library in (a) is performed.
  • a 5 'UTR sequence amplification primer sequence-SP6 promoter sequence- ⁇ 29 enhancer sequence
  • a 5 'UTR sequence amplification primer sequence-SP6 promoter sequence- ⁇ 29 enhancer sequence
  • a 5 'UTR sequence amplification primer sequence-SP6 promoter sequence- ⁇ 29 enhancer sequence
  • a 5 'UTR sequence amplification primer sequence-SP6 promoter sequence- ⁇ 29 enhancer sequence
  • a T7 tag MASMTGGQQMG (SEQ ID NO: 118)
  • This tag may be any commonly used tag.
  • FLAG tag (DYKDDDDK (SEQ ID NO: 119))
  • STOP codon, insertion, deletion some undesired mutations (STOP codon, insertion, deletion) by PCR amplification, chemical synthesis, etc. Therefore, the C-terminal tag allows you to select only in-frame tags, which can also be used with any commonly used tag. Absent.
  • the protein is a single chain antibody.
  • H chain and L chain of mouse spleen-derived antibody (IgG) mRNA These variable regions were extracted by RT-PCR, and the C-terminus of the H chain and the N-terminus of the L chain were
  • FIG. 2 shows an outline of the steps of the selection method of the present invention when a single-chain antibody DNA library is used.
  • the desired antibody is enriched by such a selection cycle, ie, by repeating selection, amplification, library restructuring, and translation.
  • a certain selection pressure at the time of selection the properties of the obtained antibody and the concentration efficiency are determined.
  • the target antibody can be selected even when a high selection pressure is applied so that the target antibody cannot be obtained by the conventional method.
  • the coding part of the protein to be selected comprises a 5 'terminal region, an ORF region, and a 3' terminal region, and may or may not have a Cap structure at the 5 'terminal.
  • the 3 'terminal region of the coding portion is a poly Ax8 sequence as an A sequence, an Xhol sequence as an X sequence, a sequence having (C or G) NN (C or G) with 4 or more bases, and an A sequence.
  • a configuration comprising an A sequence, an X sequence, or a Flag-tag sequence as an affinity tag sequence upstream of the XA sequence can be considered.
  • the affinity tag sequence is a sequence using any means capable of detecting or purifying a protein, such as one utilizing an antigen-antibody reaction such as HA-tag or IgG protein A (z domain) or His-tag. But I can't help.
  • the combination of XA sequences is important.
  • the base is important, and one having a (C or G) NN (C or G) sequence is preferred.
  • the 5 'terminal region is composed of a transcription promoter and a translation enhancer.Transcription promoters are T7 / T3, SP6, etc. can be used, and there is no particular limitation!
  • a part of the omega sequence (029) of the translation enhancer includes a part of the omega sequence of TMV (Gallie DR, Walbot V. (1992) Nucleic Acids Res., 20, 4631-4638).
  • the polyethylene glycol (PEG) portion is a CCA region, a PEG region, and a donor region.
  • the minimum required configuration is the donor region. From the viewpoint of translation efficiency, those having a PEG moiety as well as the donor region are more preferable, and those having a pure mouth mycin which has no binding ability to amino acids are preferable!
  • the molecular weight range of the polyethylene glycol in the PEG region is from 400 to 30,000, preferably from 1,000 to 10,000, and more preferably from 2,000 to 6,000.
  • the CCA domain can be configured with or without Puromycin!
  • a configuration containing puromycin is used in a cell-free translation system so that a spacer can bind to the C-terminus of the protein translated there.
  • Including puromycin includes including derivatives thereof. Examples of derivatives include the following. 3 N-aminoacylpuromycin aminonucleosides (3'- ⁇ —Aminoacylpuromycin aminonucleoside, PANS-amino acids), such as PANS-Gly for glycine in the amino acid portion, PANS-Val for phosphorus, PANS-Ala for alanine, etc. PANS-all amino acids corresponding to all amino acids can be used.
  • 3'-N-aminoacyl adenosine aminonucleoside an amide bond formed as a result of dehydration condensation of the amino group of 3'-aminoadenosine and the carboxyl group of amino acid as a chemical bond
  • AANS-amino acid 3'-Aminoacyladenosine aminonucleoside
  • AANS-amino acid for example, AANS-Gly of glycine, AANS-Val of norin, AANS-Ala of alanine, and other AANS-amino acids corresponding to all amino acids
  • nucleosides or those in which a nucleoside and an amino acid are ester-linked can also be used.
  • Other substances that have a chemical structural skeleton similar to nucleosides or nucleosides Any substance can be used as long as the substance has a chemical structure and can be chemically bonded.
  • CCA CCA region
  • a structure containing no puromycin or a structure containing puromycin having no binding ability to amino acids is preferable. Any substance in which the amino group of the above puromycin derivative lacks the ability to bind to amino acids, and the CCA region lacking puromycin may also be a potential force.Includes puromycin, which cannot bind proteins on the ribosome. Efficiency can be increased. The reason is unclear, but puromycin, which cannot bind to proteins, may stimulate turnover by stimulating liposomes. In the case of uncombined piuromycin, the above puromycin is made uncombinable with amino acids in an appropriate manner.
  • the PEG portion can be configured to have a modifying substance.
  • the translation template can be recovered, reused by purification, or used as a tag for fixing.
  • a modifying substance a fluorescent substance, biotin, or various separation tags such as His-tag may be introduced into at least one base of DNA and / or RNA.
  • each length is about 60 bp in the 5 'end region. It is about 40 bp in the 3 'end region, and has a length that can be designed as an adapter region in a PCR primer. This has created a new effect.
  • any vector, plasmid or cDNA library PCR can easily create a coding part having a 5'-terminal region and a 3'-terminal region.
  • a translation template with high translation efficiency can be obtained.
  • the ligation of the PEG part and the code part is performed by a general DNA ligase method.
  • the method is not particularly limited, and any method such as a method using a compound or a photoreaction can be used.
  • the A sequence in the 3'-terminal region is important as the range that affects the ligation efficiency in the coding region, and at least 2 residues of dA and / or rA mixed or single It is a poly A continuous chain, preferably a poly A continuous chain of 3 or more residues, more preferably 6 to 8 residues or more.
  • the DNA and / or RNA sequence at the 5 'end of the donor region of the PEG part affects ligation efficiency.
  • dC dihydroxycytidylic acid
  • dCdC diideoxycytidylic acid
  • the type of the base is preferably in the order of C> (U or T)>G> A. Further, it is preferable to add polyethylene glycol having the same molecular weight as the PEG region during the ligation reaction.
  • the protein to be selected which is synthesized by the translation of the protein to be selected with the translation template in the presence of the modifying agent, and is C-terminally modified with the modifying agent, and also serves as the translation template and the modifying agent.
  • the feature lies particularly in the configuration of the code part of the translation template. The details are described below.
  • the PEG portion of the translation template is characterized in that puromycin cannot be linked to amino acids.
  • the 3 ′ terminal region is an XA sequence, and the first 4 bases are important in the X sequence, and (C or G ) Those having the sequence of NN (C or G) are preferred.
  • the length is about 60 bp and about 40 bp in the 3'-terminal region, and it can be designed as an adapter region for PCR primers. This makes it possible to easily create a code portion having a 5'-terminal region and a 3'-terminal region by PCR, and ligating a PEG portion instead of the 3'UTR to this code portion to obtain a C-terminal portion.
  • a translation template with high translation efficiency suitable for labeling can be obtained.
  • the modifying agent is an axepept having a group (including a residue) capable of binding to a protein by a transpeptidation reaction in a protein translation system, ie, a transpeptidation reaction on a ribosome.
  • the linker has a configuration in which the linker is linked to the modifier via a nucleotide linker.
  • the fluorescent substance examples include fluorescent dyes such as fluorescein series, rhodamine series, Cy3, Cy5, eosin series, and NBD series, and fluorescent proteins such as green fluorescent protein (GFP).
  • any compound may be used as a marker, such as a coenzyme such as biotin, a protein, a peptide, a saccharide, a lipid, a dye, and polyethylene glycol.
  • the modifying portion preferably has a fluorescent group, a group that binds to a protein, or both.
  • the receptor section has a group capable of binding to a protein by a transpeptidation reaction in a protein translation system, and preferably has a residue of pure mouth mycin or a derivative thereof.
  • Puromycin has a structure similar to aminoacyl-tRNA and is known as an antibiotic that inhibits protein synthesis, but is known to bind to the C-terminus of proteins at low concentrations (Miyamoto-Sato, E. et. al. (2000) Nucleic Acids Res. 28: 1176-1182).
  • the puromycin derivative that can be used is any substance having a structure similar to puromycin and capable of binding to the C-terminus of the protein.
  • nucleotide linker that connects between the modification part and the receptor part is, specifically, a nucleic acid or a nucleic acid derivative in which one or more ribonucleotides or deoxyribonucleotides are connected, and particularly preferred examples include:
  • An example is a conjugate having one ribonucleotide (-rC-) or one deoxyribonucleotide (_dC-) containing a cytosine base.
  • the nucleotide linker is deoxycytidylic acid, 2'-deoxycitidyl- (3 ', 5')-2'-deoxytidylic acid, ribocytidylic acid, or ribocytidyl- (3 ', 5')- Preferably it is ribocytidylic acid! / ,.
  • the modifying agent can be produced by binding the above-mentioned modified portion and the acceptor portion via a desired nucleotide linker by a chemical bonding method known per se.
  • the above-mentioned receptor portion protected with an appropriate protecting group is bound to a solid-phase carrier, and a nucleotide phosphoramidite and a deoxynucleotide phosphoramidite are used as a nucleotide linker using a nucleic acid synthesizer. It can be produced by sequentially binding a phosphoramidite to which a fluorescent substance or biotin is bound as a functional modifier, and then performing deprotection.
  • the above components or the type of bonding can be combined by a liquid phase synthesis method, or both can be used in combination.
  • a metal ion such as nickel
  • the metal ion can be coordinated with a chelating reagent such as triacetate triacetic acid or iminodiacetic acid, and then the metal ion is distributed in the next step. Can be placed.
  • the PEG portion of the translation template is the same as described above, except that puromycin can be linked to amino acids.
  • the coding portion is the same as that described above.
  • the 3 ′ end region is an A sequence, and the efficiency of association of total proteins is significantly improved. It was confirmed that the amount of free protein was drastically reduced.
  • PEG-C-terminally modified proteins can be used with RNase A when the coding part is not used for protein detection or interaction detection, for example, when applied to FCCS measurement, fluorescent readers, protein chips, etc. It is preferable to cut intentionally. Cleavage can eliminate the difficulty of detecting protein-protein interactions due to interference with the coding region.
  • a cell-free translation system In order to form IW from the protein cDNA library, a cell-free translation system is used. Specifically, thiolation such as dithiothreitol (DTT) and ⁇ -mercaptoethanol A wheat germ extract containing or not containing the compound, a heron reticulocyte extract, and an Escherichia coli S-30 extract are used. In particular, it has been difficult to express an active antibody as a protein in the cell-free translation system containing DTT, but in the present invention, it is possible to select an active antibody by using IW even in the presence of DTT. I was able to do it.
  • DTT dithiothreitol
  • ⁇ -mercaptoethanol A wheat germ extract containing or not containing the compound, a heron reticulocyte extract, and an Escherichia coli S-30 extract are used.
  • DTT dithiothreitol
  • ⁇ -mercaptoethanol A wheat germ extract containing or not containing the compound,
  • the translation template of the above protein is caloried, and incubated at 25-37 ° C for several hours to form IW.
  • a C-terminal modified protein is synthesized.
  • the synthesized IVV is directly used for the next heat treatment step or reverse transcription step.
  • the assigning molecule also referred to as “IW”
  • the cell-free translation system contains a substance that inhibits the reverse transcription reaction.
  • gel filtration preferably Sephadex G200 (manufactured by Amersham Bioscience), or spin column, preferably Ultrafree MC, 100,000 NMWL (manufactured by Millipore), or PURESYSTEM (post-genome) of E. coli cell-free translation system (Manufactured by Research Institute), use nickel resin.
  • RNA-DNA hybrid By making the mRNA portion of the IW into an RNA-DNA hybrid by the reverse transcription reaction (RT), nonspecific adsorption of the mRNA portion to the carrier and the antigen can be prevented.
  • RT reverse transcription reaction
  • mRNA is denatured by heating at 65 ° C, then cooled to 4 ° C to anneal, ReverTra Ace (TOYOBO) is added, and the reaction is performed at 50 ° C for 30 minutes.
  • the reverse transcriptase may be any enzyme under any conditions as long as it performs a reverse transcription reaction. It is not limited to the above conditions.
  • the heat treatment conditions of IVV with mRNA part as RNA-DNA hybrid are also usually in the range of 1 to 30 minutes at 50-100 ° C. Force that is the condition selected is higher than the maximum temperature of reverse transcription. .
  • the DNA polymerase used for RT-PCR and PCR is not particularly limited as long as it is used for the PCR reaction, but the high amplification efficiency and the fidelity of the PCR are not too high. Are more preferred.
  • the KOD Dash (TOYOBO product) used in the present invention can be efficiently amplified even from a very small amount of type I DNA, and can be evolved into a more highly functional one because appropriate mutations are introduced.
  • the target molecule is a peptide (which may be chemically synthesized or isolated from nature or may be a partial digest of a protein), a protein, a nucleic acid (DNA or RNA), a saccharide, various small molecules. All compounds and substances, such as compounds, metals and metal compounds, are applicable.
  • the target molecule used in the present invention may be a force capable of binding to a solid phase such as a resin or a bead.
  • a solid phase such as a resin or a bead.
  • One that is bound by an outer portion is included.
  • the modifying substance used in the case of binding via the modifying substance is usually a molecule that specifically binds to a specific polypeptide (hereinafter, may be referred to as "ligand”).
  • a specific polypeptide hereinafter, sometimes referred to as “adaptor protein” that binds to the ligand is bound to the phase surface.
  • the adapter protein also includes a binding protein, a receptor protein constituting the receptor, an antibody, and the like.
  • adapter protein Z ligands examples include biotin-binding protein Z-biotin such as avidin and streptavidin, maltose-binding protein Z-maltose, G-protein Z guanine nucleotide, polyhistidine peptide Z-nickel, and cobalt.
  • biotin-binding protein Z-biotin such as avidin and streptavidin
  • maltose-binding protein Z-maltose G-protein Z guanine nucleotide
  • polyhistidine peptide Z-nickel examples of the combination of adapter protein Z ligands include biotin-binding protein Z-biotin such as avidin and streptavidin, maltose-binding protein Z-maltose, G-protein Z guanine nucleotide, polyhistidine peptide Z-nickel, and cobalt.
  • Various receptors such as metal ion, glutathione S-transferase Z glutathione
  • combinations of adapter protein Z ligands include a biotin binding protein such as avidin and streptavidin, a maltose binding protein Z maltose, a polyhistidine peptide Z a metal ion such as nickel or cobalt, and glutathione. Particularly preferred is a combination of streptavidin z-biotin, in which s-transferase Z daltathione is preferred.
  • the binding of the adapter protein to the surface of the solid phase can be carried out by a method known per se, and specifically, for example, tannic acid, formalin, dartalaldehyde, pyrvicaldehyde, benzodiazobenzidine , Toluene-2,4-diisocyanate, amino group, carboxyl group convertible to active ester, or water convertible to phosphoramidite
  • a method using an acid group or an amino group can be used.
  • a known method usually used to bind proteins, nucleic acids, sugar chains, and low molecular weight compounds to the solid phase specifically, for example, tannic acid , Formalin, glutaraldehyde, pyrvicaldehyde, bis-diazodyl benzidine, toluene-2,4-diisocyanate, amino group, hydroxyl group which can be converted to active ester, or hydroxyl or amino group which can be converted to phosphoamidide Can be used.
  • the solid phase carrier is preferably agarose beads or agarose beads in which a magnetic substance is embedded.
  • the distance between the solid surface and the antigen molecule should be more than 30 angstroms from a steric point of view!
  • a high affinity antibody There are generally two methods for obtaining a highly functional protein.
  • a high affinity antibody will be described as an example.
  • One is to set the antigen concentration to be low in the selection experiment and select only the antibody at that concentration.
  • the amount of antibody to be recovered is naturally small, and the efficiency is low. Accordingly, the antigen concentration that can be set is limited to the concentration that can be recovered.
  • the second is off-rate selection (Hawkins, R. (1992) J. Mol. Biol. 226, 889-896; Boder, E. (1997) Nat. Biotechnol. 15, 553-557; Jermutus, L. (2001 Natl. Acad. Sci. USA 98, 75-80), which uses the dissociation rate of antigen and antibody.
  • dissociation constant (Kd) is represented by the ratio of the association rate constant (on-rate) and dissociation rate constant (off-rate), association constant of biopolymers substantially within a certain range (10 4 - 10 6 M — Because of the ⁇ , the real Kd is determined by the dissociation rate constant, so antibodies with a slower dissociation rate have a higher affinity, so the longer the elution time with the antigen, the more concentrated the antibody with higher affinity
  • this method has the disadvantage that it takes longer to perform one cycle of a selection experiment, and a solution to this problem is to use an antigen or antibody sequence-specific instead of eluting with the antigen.
  • protease is eluted by quenching with proteinase, which has a very high substrate specificity, such as TMV protease factor Xa, from trypsin, proteinase K, and the like. Specificity The above methods are also applicable to the present invention, and are not limited by any proteases including, but not limited to, Is an extremely effective method.
  • a method for producing a protein using the nucleic acid selected by the selection method of the present invention that is, a step of selecting a nucleic acid encoding a protein that interacts with a target molecule by the selection method of the present invention, and A method for producing a protein by translating a selected nucleic acid is also provided.
  • the step of selecting a nucleic acid is as described for the selection method of the present invention.
  • the nucleic acid may be translated by a cell-free translation system, or a living cell such as Escherichia coli may be transformed with the nucleic acid by using a plasmid into which the nucleic acid has been introduced, and the protein may be expressed in the living cell. You may let them.
  • a cell-free translation system a cell-free translation system containing a thiol compound such as dithiothreitol or j8-mercaptoethanol, preferably, a wheat germ extract, a rabbit egret reticulocyte extract, or an Escherichia coli S-30 extract Liquid.
  • the protein When expressing the protein, the protein may be expressed as a fusion protein of a protein encoded by the selected nucleic acid and an enzyme or a green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • Examples of the protein obtained by the production method of the present invention include a single-chain antibody.
  • a single-chain antibody having angiotensin II binding activity which has the amino acid sequence shown in the following (A) or (B).
  • SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 or Pamino 105 is an amino acid rooster.
  • a single-chain antibody having binding activity to Lewis X having the amino acid sequence shown in the following (A) or (B).
  • Cell-free translation systems include wheat germ extracts, egret reticulocyte extracts, and E. coli S-30 extracts.
  • Specific examples of expressing proteins in large amounts using living cells include bacteria such as Escherichia coli, Bacillus subtilis, thermophiles, and yeast, cultured cells such as insect cells and mammals, as well as nematodes, Drosophila, Any cells, such as zebrafish and mice, may be used.
  • bacteria such as Escherichia coli, Bacillus subtilis, thermophiles, and yeast
  • cultured cells such as insect cells and mammals, as well as nematodes, Drosophila, Any cells, such as zebrafish and mice, may be used.
  • the above-mentioned C-terminal labeled ⁇ can directly introduce both of the associated modified proteins, or, if the above-described translation template is introduced and the C-terminal labeling is carried out, simultaneously.
  • a modified protein is synthesized by introducing a 100 / zM modifier into cells by electroporation, microinjection, or the like, and keeping the cells at the optimal growth temperature for several hours.
  • mapping the mapping molecule is synthesized by introducing the above translation template and keeping the cells at the optimal growth temperature for several hours. Both synthesized modified proteins can be recovered by disrupting the cells and subjected to subsequent purification or detection processes. In addition, subject to the detection process in the cell as it is Is also possible. Select an appropriate translation template according to the translation system used
  • RNA promoters are used for transcription into RNA. Since the protein to be expressed may be toxic to the host Escherichia coli, most vectors have some form of expression control. This control is also important so that the growth of E. coli is not hindered until E. coli is transformed with a vector containing the gene encoding the expression protein and expression is induced.
  • a ribosome binding site Immediately downstream of the promoter and immediately upstream of the claw site, a ribosome binding site,
  • the higher-order structure of mRNA near the translation start point is important.
  • the expression system in Escherichia coli is performed in the cytoplasm.
  • proteins having an SS bond, such as antibodies become inclusion bodies. Therefore, in the case of an antibody, E. coli expression is generally performed in periplasm under an irritating environment. In this case, it is achieved by introducing a signal sequence (for example, pel B leader or ompT) into the N-terminus of the antibody.
  • a signal sequence for example, pel B leader or ompT
  • the expression level in periplasm is extremely low compared to the cytoplasm.
  • DTT exists in the antibody obtained in the present invention. Since it has been selected under a reducing environment, it can be expressed as a water-soluble functional antibody even when expressed in the cytoplasm of Escherichia coli.
  • the single-chain antibody obtained by the production method of the present invention can be used for immunological detection of a protein.
  • the immunological detection method include a Western blot method, an immunostaining method, a fluorescent antibody staining method, an antibody chip method, and an immunoprecipitation method.
  • the protein interaction is detected by bringing the single-chain antibody obtained by the production method of the present invention into contact with a protein and detecting the interaction between the single-chain antibody and the protein.
  • Methods for detecting protein interactions include fluorescence correlation spectroscopy, fluorescence imaging analysis, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, and enzyme-linked immunosorbent assay.
  • the single-chain antibody, the peptide, and the protein are modified with some modifying agent, for example, a fluorescent dye, or an enzyme or a fluorescent protein ( It is necessary to construct a fusion protein with GFP) and detect it.
  • some modifying agent for example, a fluorescent dye, or an enzyme or a fluorescent protein
  • the method for modifying single-chain antibodies, peptides and proteins with fluorescent dyes has been described above. Construction of the fusion protein involves fusing GFP or an enzyme, preferably alkaline phosphatase ⁇ horseradish rust) peroxidase, to the C-terminus or N-terminus of the single-chain antibody.
  • Such a single-chain antibody fusion protein is obtained by expression and purification in a cell-free translation system or a system of Escherichia coli, baculovirus, or animal cells. Fluorescence is detected for GFP, visible light for alkaline phosphatase, and chemiluminescence for horseradish peroxidase with a luminometer.
  • a fusion protein of a secondary antibody of a single-chain antibody tag Flag-tag, T7-tag, HA-tag, etc.
  • GFP or alkaline phosphatase ⁇ horseradish peroxidase is used. May be.
  • tissue staining using antibodies is relatively simple in operation, and only an antibody that recognizes the target molecule is prepared.
  • antibodies that recognize that molecule are often obtained in some way, so even the first person can start the experiment immediately.
  • Another advantage of the present method is that the presence of a protein can be directly confirmed in the form of gene expression.
  • not all antibodies can be used for tissue staining. Good results cannot be expected with antibodies because they cannot recognize the structure of the molecule in vivo. The determination of the ability to prepare antibodies suitable for tissue staining is a key game.
  • the basic principle is the same as that of the Western plot, but in the case of tissue staining, the protein is fixed in the tissue with formaldehyde or the like, and its localization is detected.
  • Ability to detect immobilized target protein using primary and secondary antibodies If the signal is weak and difficult to detect, a method has been developed to specifically amplify only the target signal. ing.
  • observing the localization of endogenous proteins in cells using an antibody that specifically recognizes that molecule can provide very useful information. Many,. Unlike when expressed as a GFP fusion protein, etc., it is not possible to observe the movement of the molecule as it is in a living cell, but the localization of the target protein in various states such as response to stimulation and treatment of drugs and cell motility. You can observe the change in location. Even when there is no antibody against the protein of interest, localization of the molecule of interest using the antibody against the tag can be achieved by using a method such as transiently expressing the protein in a cell with the tag attached. Can be observed.
  • the localization of the tagged protein observed in this case does not always coincide with the actual localization of the protein, and is often the result of overexpression.
  • the operation of the experiment is relatively easy, it is necessary to examine the conditions of the immobilization method depending on the localization of the antibody and the target protein, and above all, prepare a specific antibody against the protein of interest Is an important point.
  • the membrane is permealized (permealized; increased permeability) using a surfactant or the like, and a specific antibody (primary antibody) against the target molecule is used.
  • a complex of the target protein and antibody is formed in the cells.
  • non-specifically bound antibodies Since many non-specifically bound antibodies remain in the cells, they are removed by washing, and then an antibody that recognizes the primary antibody and is labeled with a fluorescent substance (secondary antibody) A complex of protein-primary antibody-secondary antibody is formed. Like the primary antibody, the non-specifically bound secondary antibody is washed away, encapsulated, and observed under a microscope.
  • the Western plot refers to an operation of electrically transferring a protein after electrophoresis to a membrane, or a series of experiments including such an operation.
  • a commonly used method for protein electrophoresis is SDS-PAGE.
  • SDS as a reducing agent and 2-mercaptoethanol having a negative charge and heat-treated
  • the higher order structure of the protein is almost completely destroyed.
  • SDS binds to the protein at a protein molecular weight ratio (1: 1.4)
  • the charge state becomes uniform.
  • this is electrophoresed in a polyacrylamide gel, it can be separated according to the molecular weight of the protein.
  • the protein developed in the gel is electrophoretically transferred to a membrane, and reacted with an antibody specific to the target protein in the sample on the membrane to immunologically target the target protein. To be detected.
  • the method of Western blot using SDS-PAGE and enzyme activity is described, but it is not particularly limited to this protocol.
  • the flow of the Western blot operation is shown below. (1) After denaturing the protein sample with a sample buffer containing SDS, etc., separate proteins according to molecular weight by SDS-PAGE. (2) After electrophoresis, the gel is placed on the transfer device with the gel layered on it, and the protein band developed in the gel Is electrically transferred (plotted) onto the membrane. (3) After blocking to prevent nonspecific adsorption to the protein, a primary antibody reaction is performed with an antibody that specifically recognizes the target protein.
  • the secondary antibody reacts with a secondary antibody (with a chromogenic enzyme) that specifically recognizes the primary antibody molecule.
  • a color reaction is performed using the enzyme activity of the chromogenic enzyme attached to the secondary antibody, for example, a peroxidase activity or an alkaline phosphatase activity, and the target protein is detected.
  • Immunoprecipitation refers to a specific antigen-antibody reaction and modification of the C-terminal portion of a single-chain antibody with biotin, various tags such as Flag-tag, T7-tag, By using HA-tag, His-tag, etc. (this binds to streptavidin or tag antibody covalently bound to agarose beads, it can be easily separated from the solution by centrifugation), and the antigen corresponding to the used antibody can be obtained. It is a method to separate and purify only proteins. However, at present, not only is the antigen protein separated and purified, but rather the complex with the antigen protein that directly binds to the antibody by appropriately weakening the washing strength and the lysate detergent used.
  • the high-affinity antibodies selected in the present invention serve the purpose. Commonly used antibodies require higher titers (10 7 -IOVOI 1 ) than Western blots, and require a certain degree of specificity.
  • the aspect of the protein complex that can be separated changes depending on the buffer used for washing, the number of washings, the composition of the solubilization buffer, and the like.
  • the protocol is shown below, but is not particularly limited.
  • (1) Recovery of lysate As a preparation step for performing immunoprecipitation, it is necessary to first obtain a cell-free protein solution from cells. Depending on the concentration and composition of the solution, the results of subsequent immunoprecipitation will also vary. The lysate contains various proteins and various proteins.
  • Antigen-antibody reaction An antigen-antibody reaction is caused by adding a single-chain antibody that recognizes the target protein to the lysate.
  • the modification obtained above is usually used.
  • the functional protein and the target molecule are brought into contact with each other by appropriately combining them according to the type of the modifying substance, the type of the reaction system, etc., and the signal generated by the modified functional protein or the target molecule is based on the interaction between the two molecules. ! / Analyze the interaction by measuring the change in the signal generated.
  • the analysis of the interaction may be performed, for example, by fluorescence correlation spectroscopy, fluorescence imaging analysis, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, or solid-phase enzyme immunoassay. It is done by law. Further, the two sets of the modified functional protein obtained above and the target molecule are brought into contact with each other by appropriately combining them according to the type of the modifying substance, the type of the reaction system, and the like. The interaction is analyzed by measuring a change in the signal generated based on the interaction between the antigen molecules.
  • Interaction analysis can be performed, for example, by fluorescence correlation spectroscopy, fluorescence imaging analysis, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, or enzyme-linked immunosorbent assay. Is performed by The details of these methods are described below. The target molecules and interactions are as described above.
  • the functional protein to be used may be used after being modified with a modifying substance according to the embodiment. Can do.
  • the modifier is usually selected from non-radioactive modifiers such as fluorescent substances.
  • the fluorescent substance various fluorescent dyes having free functional groups (for example, carboxyl group, hydroxyl group, amino group, etc.) and capable of being linked to the above-mentioned single-chain antibodies such as proteins and nucleic acids, for example, fluorescein series, rhodamine series, Any of Cy3, Cy5, eosin series, NBD series and the like may be used.
  • the type and size of the compound are not limited as long as the compound can be modified such as a dye.
  • these modifying substances those suitable for a method of measuring or analyzing a change in a signal generated based on an interaction between a target molecule and a modified functional protein are appropriately used.
  • the above-mentioned modifying substance can be bound to the functional protein using an appropriate method known per se. Specifically, for example, the above-described method of modifying the C-terminus can be used.
  • the modified functional protein or the target molecule used in the present invention may be bound to a solid phase depending on the embodiment. Those that are combined by other parts are listed.
  • the modifying substance used in the case of binding via a modifying substance is usually a molecule that specifically binds to a specific polypeptide (hereinafter, may be referred to as "ligand”).
  • a specific polypeptide hereinafter, sometimes referred to as “adaptor protein” that binds to the ligand is bound to the phase surface.
  • the adapter protein also includes a binding protein, a receptor protein constituting the receptor, an antibody, and the like.
  • adapter protein Z ligands examples include biotin-binding protein Z-biotin such as avidin and streptavidin, maltose-binding protein Z-maltose, G-protein Z guanine nucleotide, polyhistidine peptide Z-nickel, and cobalt.
  • biotin-binding protein Z-biotin such as avidin and streptavidin
  • maltose-binding protein Z-maltose G-protein Z guanine nucleotide
  • polyhistidine peptide Z-nickel examples of the combination of adapter protein Z ligands include biotin-binding protein Z-biotin such as avidin and streptavidin, maltose-binding protein Z-maltose, G-protein Z guanine nucleotide, polyhistidine peptide Z-nickel, and cobalt.
  • Various receptors such as metal ion, glutathione S-transferase Z glutathione
  • adapter protein Z ligands include biotin-binding proteins such as avidin and streptavidin, and maltose-binding protein Z-matrix. Particularly preferred is a combination of streptavidin Z-biotin, which is preferably a metal ion such as lactose, polyhistidine peptide Z nickel or cobalt, glutathione s-transferase Z daltathione, and the like. These binding proteins are known per se, and the DNA encoding the protein has already been closed.
  • the binding of the adapter protein to the solid phase surface can be carried out by a method known per se, and specifically, for example, tannic acid, formalin, dartalaldehyde, pyrvicaldehyde, benzodiazobenzidine
  • a method utilizing a toluene-2,4-diisocyanate, an amino group, a carboxyl group convertible to an active ester, or a hydroxyl group or amino group convertible to a phosphoramidide can be used.
  • a known method usually used for binding proteins, nucleic acids, sugar chains, and low molecular weight compounds to a solid phase specifically, for example, tannic acid , Formalin, glutaraldehyde, pyrvicaldehyde, bis-diazoi di-benzizone, toluene-2,4-diisocyanate, amino group, hydroxyl group that can be converted to active ester, or hydroxyl group or amino group that can be converted to phosphoamidide
  • a method can be used.
  • Measurement is a means for collecting changes in signals used for analysis, and should not be construed as limiting in any sense.
  • Examples of the measuring method used include fluorescence correlation spectroscopy, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, fluorescence imaging analysis, surface plasmon resonance, and enzyme-linked immunosorbent assay. Any system that can detect intermolecular interactions is available.
  • Proc. Natl. Acad. Sci. USA, 91, 5740-5747 (1994)) is a method for measuring the flow velocity, diffusivity, volume contraction, etc. of particles under a confocal laser microscope or the like.
  • the interaction between the modified functional protein (C-terminal modified functional protein) and the antigen molecule is measured by measuring the change in the translational Brownian motion of one original modified molecule, thereby measuring the interacting molecules. can do.
  • the sample particles are excited by the excitation light, emit fluorescence in a part of the sample solution volume, and the emitted light is measured and Get the percentage. This value varies with the number of particles present in the volume of space observed at a particular time.
  • FCCS fluorescence cross-correlation spectroscopy
  • FRET fluorescence resonance energy transfer
  • the FCCS method when compared with other detection systems such as the fluorescence depolarization method, the FCCS method has advantages such as a small sample amount required, a short detection time, and easy automation for HTS. Furthermore, the FCCS method can provide very basic information such as the size and number of fluorescently labeled molecules, and may be used for general-purpose applications such as surface plasmon resonance. The difference between the two is that the surface plasmon resonance method detects the interaction in a state where the protein is immobilized, whereas the FCCS method allows the interaction in a solution that is closer to the natural state to be observed. is there. In the FCCS method, instead of the necessity of immobilizing proteins, it is necessary to label proteins with fluorescent dyes. The present invention has made it possible to overcome this problem.
  • a method for bringing the two into contact with each other is sufficient to allow the two target molecules to interact with each other. Any method may be used as long as it comes into contact with the substrate, but preferably, the emission wavelengths do not overlap at a concentration appropriate for a buffer or the like normally used for biochemistry in a measuring well of a commercially available FCCS device.
  • a solution in which two types of C-terminal modified functional proteins labeled with a fluorescent dye are dissolved, and a solution in which two unlabeled target molecules are dissolved in the same buffer at an appropriate concentration are added. Be done.
  • a method of performing a large number of analyzes at the same time for example, a plurality of different C-terminal modified functional proteins are respectively injected into each measurement well of the above-described measurement device for FCCS, and the function of each protein is added to each.
  • a method is used in which a target molecule solution capable of binding to a functional protein is charged, or a specific C-terminal modified functional protein is charged, and a plurality of different target molecule solutions are charged into each well.
  • a modified molecule is brought into contact with a solid-phased molecule, and emitted from the modified molecule remaining on the solid-phased molecule due to the interaction between both molecules.
  • This is a method of measuring or analyzing fluorescence using a commercially available fluorescence imaging analyzer.
  • one of the C-terminal modified functional protein and the target molecule may be immobilized by the method described above. Needs to be When the target molecule is used in the form of a solid phase, it can be modified, and can be used. In addition, when used without being immobilized, it is necessary to be modified with the above-mentioned modifying substance.
  • the C-terminal modified functional protein even if it is immobilized via the modified part, is immobilized with a part other than the modified part, for example, a fusion protein such as GST, Flag-tag, His-tag, etc. Can also be used.
  • a substrate for immobilizing a C-terminal modified functional protein or a target molecule is usually used for immobilizing proteins, nucleic acids, or the like-trocellulose membrane, nylon membrane, slide glass, or the like. Plastic microplates can also be used.
  • a modified target molecule ! a method for bringing a functional protein into contact with a solid-phased molecule as long as the two molecules can be brought into contact with each other to an extent sufficient for interaction. It is possible to use a modified target molecule! /, To prepare a solution in which a C-terminal modified functional protein is dissolved at an appropriate concentration in a buffer commonly used in biochemistry. However, a method of bringing this into contact with the surface of the solid phase is preferred.
  • a step of washing the excessively present modified target molecule or C-terminal modified functional protein with the same buffer or the like is carried out, and the target molecule or c-terminal remaining on the solid phase is removed.
  • a fluorescent signal that also emits the power of the modifying substance of the terminally modified functional protein, or a signal in which the fluorescent light emitted by the modified molecule that is immobilized on the solid phase and the fluorescent light that is emitted from the modifying molecule that remains on the solid phase are used as a commercially available signal.
  • Molecules that interact with immobilized molecules can be identified by measurement or analysis using an imaging analyzer.
  • this method as a method for performing a large number of analyzes at the same time, for example, a method in which a plurality of c-terminal modified functional proteins or modified or unmodified target molecules are addressed and immobilized on the solid phase surface, Alternatively, a method of immobilizing one kind of C-terminal modified functional protein or a modified or unmodified target molecule on a solid phase and contacting a plurality of kinds of c-terminal modified functional proteins or modified target molecules is used. .
  • a plurality of types of C-terminal modified functional proteins or modified target molecules are brought into contact with each other, the molecules remaining on the solid phase are obtained by dissociation due to a difference in buffer concentration or the like, and this is obtained by a known method. It can be identified by analysis.
  • FRET fluorescence resonance energy transfer
  • FRET does not occur because the distance is large, and the force of observing the fluorescence spectrum of the donor
  • the fluorescence spectrum of the acceptor is observed by FRET.
  • Fluorescent spark The presence or absence of protein-protein interaction can be determined from the difference in the wavelength of the torr.
  • a fluorescent dye a combination of fluorescein as a donor and rhodamine as an acceptor is often used.
  • GFP fluorescent green protein
  • a substrate for immobilizing the C-terminal-modified functional protein or the target molecule a substrate made of a material such as glass is used, and quartz glass is preferably used. It is also preferable that the surface is ultrasonically cleaned to prevent scattering of laser light.
  • a method for bringing a C-terminal modified functional protein or a modified target molecule into contact with an immobilized molecule by immobilizing the molecule to the immobilized molecule is sufficient to allow both molecules to interact with each other. Any method may be used as long as it can be contacted with each other, but it is preferable that the non-immobilized C-terminal modified functional protein or the modified target molecule is added at a concentration appropriate for a buffer commonly used in biochemistry. A preferred method is to prepare a dissolved solution and drop it on the surface of the solid phase. [0108] After the two molecules are brought into contact, the fluorescence excited by the evanescent field illumination is measured using a detector such as a CCD camera to identify the molecules that interact with the immobilized molecules. Can be.
  • a detector such as a CCD camera
  • a method of performing a large number of analyzes at the same time for example, a method of addressing a plurality of C-terminal modified functional proteins or modified target molecules on the substrate and immobilizing the same on the substrate is used.
  • the fluorescence polarization method uses a method in which a fluorescent molecule excited by fluorescence polarization emits light during the excited state. When the steady state is maintained, fluorescence is emitted in the same plane of polarization.However, when the excited molecule performs a rotating Brownian motion or the like during the excited state, the emitted fluorescence is different from the excitation light. This is a method that utilizes the fact that it becomes a plane.
  • the motion of a molecule is affected by its size, and when the fluorescent molecule is a macromolecule, the motion of the molecule during the excited state is almost negligible, whereas the emitted light remains polarized.
  • the polarization of the emitted light is eliminated because the moving speed is high. Therefore, the intensity of the fluorescence emitted from the fluorescent molecule excited by the plane-polarized light is measured on the original plane and a plane perpendicular to the original plane. The information about is obtained.
  • the behavior of a target molecule that interacts with a fluorescence-modified molecule without being affected by contaminants can be tracked. This is also a force that is measured as a change in the degree of polarization only when the target molecule interacts with the fluorescently modified molecule.
  • BECON manufactured by Panyera
  • this method can also be performed by using these apparatuses.
  • any method of contacting the two sets of C-terminal modified functional protein target molecules can be used as long as the two molecules can be brought into contact enough to interact with each other.
  • a solution prepared by dissolving the c-terminal modified functional protein at an appropriate concentration in a buffer or the like normally used in biochemistry is added to a measuring well of a commercially available fluorescence depolarizer.
  • the method is carried out by injecting a solution in which the target molecule is dissolved at an appropriate concentration in the same buffer.
  • the degree of the interaction In order to detect the optimal combination of the interaction between two targets measured in the present method, it is effective to numerically indicate the degree of the interaction.
  • an index indicating the degree of interaction for example, a value of a minimum target substance concentration that gives a maximum fluorescence polarization degree to a certain concentration of a C-terminal modified functional protein can be used.
  • the surface plasmon resonance method is a method in which surface plasmons are excited by molecules interacting at the metal-Z liquid interface, and are measured by changes in the intensity of reflected light (Cullen, DC, et al., Biosensors). , 3 (4), 211-225 (1987-88)).
  • the C-terminal modified protein must be immobilized by the method described above, but the target molecule does not need to be modified. .
  • a transparent substrate such as glass on which a metal thin film of gold, silver, platinum or the like is formed is used.
  • the transparent substrate is generally made of glass, etc., as long as it has a material power that is transparent to laser light, regardless of what is usually used for a surface plasmon resonance device. Its thickness is about 0.1-5 mm. The appropriate thickness of the metal thin film is about 100-2000A. A commercially available solid substrate for such a surface plasmon resonance device can also be used.
  • the C-terminal modified functional protein can be immobilized on the substrate by the above-described method.
  • the method of bringing the target molecule into contact with the C-terminal-modified functional protein may be any method as long as the two molecules come into contact with each other to an extent sufficient for interaction.
  • a method can be used in which the C-terminal-modified functional protein immobilized on a solid phase is brought into contact with a solution in which a target molecule is dissolved in a buffer commonly used in biochemistry at an appropriate concentration.
  • a solid-phase enzyme immunoassay (Enzyme Linked Immunosorbent Assay (ELISA): Crowther, JR, Methods in Molecular Biology, 42 (1995)) uses a solution containing an antibody to an antigen immobilized on a solid phase. The molecules are brought into contact with each other, and the interaction (antigen-antibody reaction) between the two molecules causes the antibody remaining on the immobilized antigen to bind to the modified molecule (such as IgG) that specifically binds to the fluorescent or modified molecule.
  • This is a method of measuring or analyzing a signal emitted from a dye as a substrate using a commercially available detector (ELISA reader).
  • a plastic microplate or the like usually used for ELISA can also be used as a substrate for immobilizing a C-terminal modified functional protein serving as an antigen.
  • the method for bringing the modified target molecule to be an antibody into contact with the solid phase molecule in the present method may be any method as long as the two molecules are brought into contact with each other to an extent sufficient for interaction.
  • a method is preferred in which a solution is prepared by dissolving a molecule in an appropriate concentration in a buffer commonly used in biochemistry, and the solution is injected into a microplate.
  • a step of washing the excessively present modified molecules that are not bound to the immobilized molecules with the same buffer or the like is performed to remove the modified molecules remaining on the solid phase.
  • a molecule that interacts with the immobilized antigen molecule can be identified.
  • the target molecule which has been measured by each of the above methods and has been found to have an interaction, is identified by an appropriate method known per se when the primary structure of the molecule is unknown.
  • the primary structure can be analyzed. Specifically, when the target molecule for which interaction has been recognized is a protein, the primary structure can be identified by analyzing the amino acid sequence using an amino acid analyzer or the like. Further, when the target molecule is a nucleic acid, the base sequence can be determined by a base sequence determination method using an auto DNA sequencer or the like.
  • the above-described method for immobilizing a C-terminal modified functional protein or target molecule on a solid phase via a modified portion is used.
  • the device can be constructed by combining known suitable means. Each means in the apparatus is known, and operations such as holding a substrate, adding a C-terminal modified protein solution, and washing may be performed by a method known per se. By combining these operations, a fully automatic or semi-automatic device for immobilizing a C-terminal modified protein can be constructed.
  • an apparatus can be constructed by combining known appropriate means for performing the above-described protein-target molecular interaction measurement.
  • the respective means in the present apparatus are known, and the respective operations such as holding of the substrate, addition of antigen molecules, washing, and signal detection in these means may be performed by methods known per se. By combining these operations, a fully-automatic or semi-automatic device for protein-protein interaction measurement can be constructed.
  • High-affinity single-chain antibody selected by the method of the present invention can be used as a chimeric IgG antibody by replacing the variable region of human IgG antibody or CDR (complementarity determining region). And humanized mouse IgG antibodies. These antibodies produce little or no human anti-mouse antibody when elicited when administered to humans. Furthermore, a highly human single-chain antibody selected by the method of the present invention, which is a human antibody cDNA library, can produce a fully human monoclonal antibody by replacing the variable region of the human HgG antibody. This is an antibody that does not cause anaphylactic symptoms It can be used as a medicine.
  • a human or other animal-derived DNA library is substituted with a variable region encoded by the nucleic acid and a human IgG variable region.
  • a human or humanized antibody constructed thereby.
  • a therapeutic agent comprising the antibody as an active ingredient.
  • Antibodies that bind to angiotensin II could be used as neutralizing antibodies in the treatment of hypertension. That is, since the physiological action of angiotensin II is a blood pressure increasing action, it is considered that the antibody binds to angiotensin II and suppresses the blood pressure increasing action.
  • Antibodies that bind to Lewis X could also be used to treat cancer.
  • Lewis X When cells become cancerous, Lewis X is expressed on the cell surface, and antibodies can be used to target it.
  • an anticancer drug that kills cancer cells or a toxin such as ricin is bound and administered systemically or locally, and the cancer cells are intensively attacked like antibody-powered S missiles, resulting in cancer-specific and effective missiles Treatment can be provided.
  • an immunoribosome preparation in which a drug is put in a lipid bilayer vesicle and an antibody is bound to the surface thereof is used as a kind of preparation used for missile treatment.
  • [0131] [2] cDNA-H solution 51 synthesized in [1] 51, each HB primer (lpmol / ⁇ 1) 2.5 1 shown in the following HB primer list, 10X PCR buffer ( ⁇ ) 2.5 ⁇ l 1, MulgGl / 2 forward primer (SEQ ID NO: 48) (lpmol / ⁇ 1) 1.25 ⁇ 1, MulgG3 forward primer (SEQ ID NO: 47) (lpmol / ⁇ 1) 1.25 ⁇ 1, KOD DASH polymerase (TOYOBO) 0.25 ⁇ 1 After mixing, RNase-Free water was added to make a total volume of 25 ⁇ l, and each was subjected to PCR reaction.
  • PCR was performed at 96 ° C for 5 minutes, followed by 25 cycles of 96 ° C, 30 seconds, 50 ° C, 30 seconds, 72 ° C, and 1 minute, followed by reaction at 72 ° C for 5 minutes.
  • Amplified gene respectively check the Pando of 500-900B P by 2% Agarosugeru electrophoresis movement, Gyotsu a phenol / black port Holm process It was. That is, add the same volume of phenol: cloth form: isoamyl alcohol (25: 24: 1), mix well, centrifuge at 4 ° C for 13,200 rpm for 5 minutes, transfer only the aqueous layer to a new tube, and re-equivalent volume.
  • Phenol cloth form: isoamyl alcohol (25: 24: 1) was mixed well and centrifuged at 13,200 rpm for 5 minutes at 4 ° C, and only the aqueous layer was transferred to a new tube. The resulting solution was subjected to ethanol precipitation. That is, add a 20 mg / ml glycogen solution (Nacala Tester Co., Ltd.) 0.1 volume of 1,3 M sodium acetate (pH 5.2) and 3 volumes of 100% ethanol, leave on ice for 15 minutes, and continue at 13,200 rpm for 20 minutes.
  • a 20 mg / ml glycogen solution Nacala Tester Co., Ltd.
  • 1,3 M sodium acetate pH 5.2
  • [3] [3] 1 ⁇ l of each DNA solution (19 types) synthesized in [2], ⁇ each corresponding ⁇ ⁇ primer (lOpmol / 1) 21 shown in the primer list, 10 X PCR buffer (TOYOBO) ) 10 ⁇ 1, (2 mM) dNTPs (TOYOBO) 101, VH forward primer HF1: HF2: HF3: HF4 (1: 1: 1: 1) mixture shown in the HF primer list below (lOpmol / ⁇ ⁇ ) 2 ⁇ KOD DASH polymerase (TOYOBO) 0.5 ⁇ l was mixed, RNase-Free water was added to make a total volume of 100 ⁇ l, and each was subjected to PCR reaction.
  • PCR was performed at 96 ° C for 5 minutes, followed by 20 cycles of 96 ° C, 30 seconds, 50 ° C, 30 seconds, 72 ° C, and 1 minute, followed by a reaction at 72 ° C for 5 minutes.
  • the amplified gene was confirmed to have a band of 500-900 bp by 2% agarose gel electrophoresis, and was subjected to phenol / cloth form treatment and ethanol precipitation. Then, after centrifuging and drying for about 15 minutes, each DNA (19 species) was dissolved in 10 ⁇ l of RNase-free water.
  • [6] 5 ⁇ l of the cDNA-L solution synthesized in [5] was added to each of the LB primers (lpmol / ⁇ 1) 2.5 1, 10X PCR buffer (TOYOBO) shown in the LB primer list below.
  • 2.5 ⁇ 1, MuCK forward primer (SEQ ID No. 49) (lpmol / ⁇ 1)
  • Add 2.5 ⁇ 1, KOD DASH polymerase (TOYOBO) 0.25 ⁇ 1 add RNase-Free water, add 25 ⁇ l, and reduce the total volume to 25 ⁇
  • the PCR reaction was performed as 1 for each.
  • PCR was performed at 96 ° C for 5 minutes, followed by 25 cycles of 96 ° C, 30 seconds, 48 ° C, 30 seconds, 72 ° C, and 1 minute, followed by reaction at 72 ° C for 5 minutes. went.
  • the amplified gene was confirmed to have a band of 500-900 bp by 2% agarose gel electrophoresis, and was subjected to phenol / clonal form treatment.
  • the obtained solution was subjected to ethanol precipitation. Then, after centrifugal drying for about 15 minutes, each DNA (18 species) was dissolved in 20 ⁇ l of RNase-free water.
  • PCR was performed at 96 ° C for 5 minutes, followed by 20 cycles of 96 ° C, 30 seconds, 48 ° C, 30 seconds, 72 ° C, and 1 minute, followed by a reaction at 72 ° C for 5 minutes.
  • the amplified gene was confirmed to have a band of 500-900 bp by 2% agarose gel electrophoresis, and was subjected to phenol / cloth form treatment and ethanol precipitation. Then, after centrifugal drying for about 15 minutes, each DNA (18 kinds) was dissolved in 10 ⁇ l of RNase-free water.
  • the mixture was mixed at a ratio to obtain a total 0.5 pmol L chain DNA solution.
  • PCR was further performed to ligate one H chain and one L chain.
  • 0.5 pmol of H chain DNA solution synthesized in [4] 0.5 pmol of L chain DNA solution synthesized in [8], 5'UTR (SEQ ID NO: 57) (lpmol / 1) 0.5 ⁇ 1, McD—Linker + (SEQ ID NO: 56) (lpmol / ⁇ 1) 0.5 ⁇ 1, McD 3'UTR (His Tag) (SEQ ID NO: 55) (lpmol / ⁇ 1) 0.5 1, 10 X PCR buffer (TOYOBO) 51, 2 mM
  • PCR was performed at 96 ° C for 5 minutes, followed by 96 ° C for 30 seconds, followed by ramping to 58 ° C for 5 minutes, and 58 ° C for 30 seconds, 72 ° C for 1 minute. After 10 cycles, the reaction was performed at 72 ° C for 5 minutes.
  • PCR was performed at 96 ° C for 5 minutes, followed by 15 cycles of 96 ° C, 30 seconds, 58 ° C, 30 seconds, 72 ° C, and 1 minute, followed by reaction at 72 ° C for 5 minutes. Was done.
  • RNase-free water 100 1 was prepared, heated at 70 ° C. for 15 minutes, and then treated with TE-saturated phenol. The obtained aqueous layer was treated with phenol / chloroform, followed by ethanol precipitation using the resulting solution. The obtained pellet is centrifuged and dried for about 15 minutes,
  • RNA obtained in [13] and [12] was purified using the RNeasy Mini kit (Qiagen). That is, RNase-Free water was added to the transcription reaction solution to make the total volume 100 ⁇ l, and 350 ⁇ l of RLT buffer (Qiagen), 51 of 2-mercaptoethanol 51, and 250 ⁇ l of 100% ethanol were added, and the RNeasy mini Spin the column, centrifuge at 4 ° C, 12,000 rpm for 16 seconds, remove the discharged solution, add RPE buffer (Qiagen ⁇ OO / zl to the column, centrifuge at 4 ° C, 12,000 rpm, 16 seconds) After that, remove the discharged solution, add RPE buffer (Qiagen ⁇ OO / z 1) to the column, centrifuge at 4 ° C, 12,000 rpm for 2 minutes, remove the discharged solution, and replace the column with a new one.
  • RPE buffer Qiagen ⁇ OO / z 1
  • TOYOBO polyethylene glycol
  • PEG polyethylene glycol
  • RNA to which one spacer molecule was bound was purified using the RNeasy Mini kit (Qiagen).
  • RNA bound to one spacer molecule obtained in [14], wheat germ extract (containing 5 mM DTT) (Promega) 12.5 ⁇ 1, Amino Acid mixture, Complete (ImM) (Promega) 2 1, RNase inhibitor (TOYOBO) 2 1, CH COOK (1M) (Promega) 2 1
  • angiotensin II-biotin (0.4 M) angiotensin II-biotin or (0.4 M) Lewis X-sp-biotin dissolved in an ELISA buffer having a composition of 100 mM Tris-HC1, 150 mM NaCl, pH 7.5, 0.1% Tween 20 The mixture was rotated and stirred with a rotary mixer (Nissin) at 500 ° C for 1 hour at 25 ° C.
  • FIG. 3 shows the chemical structural formula of angiotensin II-biotin
  • FIG. 4 shows the chemical structural formula of Lewis X-sp-biotin. 5 mM biotin 71 dissolved in the ELISA buffer was added and further stirred at 25 ° C. for 30 minutes with a rotary mixer (Nissin).
  • Blocking agent Add 400 / zl of the reverse-transcribed library obtained in [18] to the suspended resin in 600 ⁇ l of the suspended resin, and use a mini disc rotor (Biocraft) at 4 ° C for 15 minutes. The rotation was stirred.
  • PCR was performed at 96 ° C for 5 minutes, followed by 25-30 cycles of 96 ° C, 30 seconds, 58 ° C, 30 seconds, 72 ° C, 1 minute, and then 72 ° C, 5 minutes. The reaction was performed. The amplified genes were confirmed to have 900-1000 bp bands by 1% agarose gel electrophoresis.
  • [0163] The amplified gene obtained in [22] was purified using Wizard Plus Minipreps DNA Purification System (Promega). That is, transfer the PCR reaction product to a 1.5 ml tube, mix 100 ⁇ l of Direct purification buffer (3 ⁇ 4r ⁇ Promega) and 1 ml of DNA purification resin (Promega), mix with 1 ml of syrup, and use a 2.5 mL syringe (Terumo) for Wizard Minicolumn (Promega). Was served. The solution was extruded with a syringe and discarded (80%), and 2 ml of isopropanol was extruded again and discarded.
  • Wizard Plus Minipreps DNA Purification System Promega
  • the concentration of the DNA solution was estimated by measuring the absorption at 260 nm.
  • PCR was performed at 96 ° C for 5 minutes, followed by 96 ° C, 30 seconds, 58 ° C, 30 seconds, 72 ° C, 1 minute for 7-10 cycles, and then at 72 ° C, 5 minutes. The reaction was performed for minutes.
  • the amplified gene was confirmed to have a band of about 1000 bp by 1% agarose gel electrophoresis, and was subjected to phenol / cloth-form treatment and ethanol precipitation. After centrifugation and drying for about 15 minutes, the DNA was dissolved in RNase-free water (101).
  • RNase-free water 100 1 was prepared, heated at 70 ° C. for 15 minutes, and then treated with TE-saturated phenol. The obtained aqueous layer was subjected to phenol / chloroform-form treatment, and then the obtained solution was subjected to ethanol precipitation. The obtained pellet was centrifuged and dried for about 15 minutes, and the DNA was dissolved in 10 ⁇ l of RNase-free water.
  • FIG. 5 shows the results obtained by subjecting each translation solution obtained in the above steps to 5% polyacrylamide electrophoresis in the presence of 8-urea to identify the associating molecules.
  • the lower bar graph shows the results of measuring and quantifying the fluorescence of FITC of the upper electrophoresis gel using MOLECULAR IMAGER FX (Bio-rad).
  • Nega is one of the clones of library MH0 obtained in [11], MH0-15
  • Posi is one of the clones of library MI3, MI3-55
  • MHO is the live one obtained in [11].
  • MHO contains a considerable proportion of proteins that are not translated into proteins due to stop codons or mutations.
  • the efficiency of force association is as low as 15%, whereas that of Mil is 25% and that of MI2 is 25%.
  • the efficiency of association has increased to 37% and 41% for MI3.
  • Nega and Posi are in-frame clones, and the association efficiency in this case is 34% and 40%, respectively.
  • MI2 or MI3 has almost the same value as the association efficiency. Therefore, the selection at the protein level was successful, indicating that the library had a higher in-frame rate! /
  • FIG. 6 shows the results obtained by subjecting each translation solution obtained in the above steps to 5% polyacrylamide electrophoresis in the presence of 8M urea in the same manner as in FIG. 5, and confirming the corresponding molecules.
  • MHO is the library obtained in [11]
  • MK1 is the library obtained in [11] at [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes
  • Lewis X is used as an antigen.
  • MK2 was prepared by reacting MK1 in [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, selecting the library using Lewis X as an antigen and recovering the library collected in [25]. Is shown.
  • a significant percentage of non-translated proteins due to stop codons or mutations contained a low efficiency of 15%, whereas 41% for MK1 and 41% for MK2. Efficiency of association increased with 36%!
  • FIG. 7 shows the results obtained by subjecting each translation solution obtained in the above steps to 5% polyacrylamide electrophoresis in the presence of 8 M urea and confirming the assigning molecules as in FIG. From the left, MM1 is the library obtained in [11], reacted at 50 ° C for 30 minutes in [18], selected angiotensin II as an antigen, and recovered in [25]. MM2 was MM1 in [18]. The library obtained in [25], MP1 was reacted with the library obtained in [11] at 99 ° C for 5 minutes at [15].
  • [18] Shows the library recovered in [25] after performing the reaction from [22] after reacting at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes.
  • the efficiency of association is 25% for MM1 and 33% for MM2.
  • MM1 has the same value as [18] compared to Mil that was reacted at 50 ° C for 30 minutes and then reacted at 99 ° C for 5 minutes at [18], and MM2 also shows an association efficiency that is inferior to MI2 This suggests that the selection at the protein level has been successful.
  • MP1 has an extremely low assignment efficiency of 8%, and it is possible that some denaturation of RNA or the assignment molecule may have occurred by reacting at 99 ° C for 5 minutes in [15].
  • FIG. 8 shows the results of PCR of the eluate obtained in [21] and PCR of the eluate obtained in [22] and 1% agarose gel electrophoresis.
  • the lower bar graph shows ethidium bromide in the upper electrophoresis gel.
  • the figure shows the results obtained by measuring and quantifying the absorption of MOLECULAR IMAGER FX (Bio-rad). From left, Nega is a library obtained in [11], MH0-15, one of the clones of MHO, Posi is a library, MI3-55, one of the clones in library MI3, and Mil is a library obtained in [11].
  • FIG. 9 shows that the flow obtained in [20] is diluted 1000-fold, the wash obtained in [20] and the eluate (Elute) obtained in [21] are diluted 1- and 10-fold. This is the result of PCR of [22] followed by 1% agarose gel electrophoresis.
  • 1 1x dilution of eluate
  • X 0.1 Abbreviation of 10-fold dilution of eluate.
  • MK1 is a library obtained by reacting the library obtained in [11] at 50 ° C for 30 minutes and then 99 ° C for 5 minutes in [18], selecting Lewis X as an antigen and collecting in [25], and MK2 is MK1. Is reacted at 50 ° C.
  • FIG. 10 shows that the flow obtained in [20] was diluted 1000-fold, and the Wash and [20] obtained in [20] were diluted.
  • the eluate obtained in [21] was diluted 1-fold and 10-fold and subjected to PCR in [22] and subjected to 1% agarose gel electrophoresis, and the absorption of ethidium bromide in the electrophoresis gel was measured.
  • [0180] [29] Colony suspension obtained in [28] 11; 10X PCR buffer (TOYOBO) 51; 2 mM dNTPs (TOYOBO) 51; M13FII (Torigami [J number 58] (10 pmol / 1) 1 1, M13RII (Toroki Column No. 59) (10 pmol / 1) 11 1, KOD DASH polymerase (TOYOBO) 0.25 ⁇ l, mix, add RNase-Free water and reduce the total volume PCR reaction was carried out with 50 ⁇ l each.
  • [0182] [30] 16 ng of DNA obtained from [29], M13FII (SEQ ID NO: 58) (1.6 pmol / ⁇ 1) 21 or M13RII (SEQ ID NO: 59) (1.6 pmol / ⁇ ⁇ ) 2 ⁇ 6 ⁇ l of DTCS kit Premix (Beckman coulter) was mixed, and RNase-Free water was added to make a total volume of 10;
  • PCR was performed at 96 ° C for 5 minutes, followed by 30 cycles of 96 ° C, 20 seconds, 58 ° C, 20 seconds, 60 ° C, and 4 minutes, followed by reaction at 72 ° C for 5 minutes. .
  • [0183] Transfer the PCR reaction product obtained in [30] to a 1.5 ml tube, and add 1 ⁇ l of 3 M NaOAc, 0.1 ⁇ EDTA 11 and 20 ⁇ g / ml glycogen solution (Nacalai Tesque, Inc.) 1 ⁇ l 1 was mixed well, and 60 ⁇ l of cold 100% ethanol was added and mixed. After centrifugation at 4 ° C and 14000 rpm for 15 minutes to remove the supernatant, washing the pellet with 70% ethanol, centrifugation again at 14000 rpm and 2 minutes to remove the supernatant was performed twice. Then, after centrifugal drying for 30 to 40 minutes, deionized formamide (Beckman coulter) 401 was added and mixed well. Sequence analysis was performed using the CEQ 2000 DNA Analysis System (Beckman coultei).
  • MHO reacted the library obtained in [11] with the library obtained in [11]
  • MHO reacted the library obtained in [11] with [18] at 50 ° C for 30 minutes, then at 99 ° C for 5 minutes, and reacted with angio.
  • the library, MI2 which selected tensin II as an antigen and recovered in [25]
  • 100 X expressed in C (%) (the number of arrays having converged arrays in A in Figs. 11 and 12) / In frame was 33% for Mil, 57% for MI2, and 87% for MI3 according to the selection.
  • 100 X in C (%) (the number of arrays with converged arrays in B in Figs. 11 and 12) / In frame shows a high value by turning the selection to 50% for MK1 and 40% for MK2.
  • the selection using Lewis X as the antigen showed that the sequence converged to the sequence shown in B.
  • 100 X (the number of arrays with converged arrays in A in Figs. 11 and 12) / In frame in C (%) is 0% for MM1 and 13% for MM2, and selection is performed once. Only by turning, the sequence shown in A was not found, and only by turning the selection twice, was it possible to find the sequence shown in A for one clone.
  • 50 ° C in [18] for MM1 and MM2 Mil, MI2, and MI3 perform the reaction at 50 ° C for 30 minutes at [18], followed by the reaction at 99 ° C for 5 minutes, while only reacting for 30 minutes.
  • Fig. 11 and Fig. 12 show the strains generated by TreeViewPPC after sequence alignment by clustalx based on the amino acid sequence, as a result of the sequence analysis and analysis performed in [31].
  • a surrounded by a line indicates the one converged by the selection of Angiotensin II
  • B surrounded by a line indicates the one converged by the selection of Lewis X.
  • the number of the clone is shown after the library abbreviation.
  • a surrounded by a line shows more than 90% homology as a result of analysis by GENETYX-MAC at the protein level compared to MI3-55, and B surrounded by a line is analyzed by GENETYX-MAC at the protein level compared to MK1-17 As a result, 90% or more homology was shown.
  • the amino acid sequence is shown as an odd SEQ ID NO: 61-117.
  • the sequence was determined as follows according to CDR sequence number i3 ⁇ 4, Martin, A. R. Accessing the Kabat Antibody Sequence Dataoase by Computer PROTEINS: Structure, Function and Genetics, 25 (1996), 130-133.
  • Each CDR of MI3-8, MK1-15, MK1-17, MK1-24, MK2-19, and MK2-8 has the following amino acid numbers.
  • PCR was performed at 94 ° C for 5 minutes, followed by 25-30 cycles of 94 ° C, 30 seconds, 58 ° C, 30 seconds, 68 ° C, 2 minutes, and then reaction at 68 ° C for 5 minutes. went.
  • the amplified genes were confirmed to have 900-1000 bp bands by 1% agarose gel electrophoresis.
  • FIG. 13 shows the results of Western blotting of [37] of ⁇ 3-55 (right two). The left three are Western blot controls. Carboxy-terminal from lane left
  • FLAG-BAP fosion protein 100 ng ⁇ 50 ng ⁇ 20 ng ⁇ Biotynylated SDS—PAGE Standards low range (Bio-rad) 21 1, MI3-55 [37] from 3rd to 6th The collected 15 1 and 51 were subjected to 15% polyacrylamide electrophoresis and transferred to a PBDF membrane.
  • Anti FLAG M2-Mouse (Sigma) was used as the primary antibody, Goat anti-mouse IgG (H + L) -HRP conjugate (Sigma) and Avidin- HRP conjugate (Bio- rad) was used as a two-following ⁇ I this ECL Western blotting detection detected in Reagents (Amersham biosciences) 7 this 1 / es Tan blot.
  • MI3-55 a band was detected at a molecular weight of about 31,000 daltons, which was consistent with the calculated value of the single-chain antibody.
  • Biacore used the Biacore 3000 system. Immobilization was performed on the sensor chip Bl (CM4) by the amine coupling method. Flow cells 1 and 2 contain 0.2 M N-ethyl- ⁇ '-dimethylaminopropylcarbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS) Activation was performed with the solution for 10 minutes. Next, 50 M streptavidin (Sigma) (0.02 M potassium phosphate buffer, pH 6.5) was mixed with 201 and 10 mM acetate buffer, pH 5.0 980 ⁇ l in flow cell 2 for 10 min. mM acetate buffer pH 5.0 was allowed to flow for 10 minutes.
  • EDC N-ethyl- ⁇ '-dimethylaminopropylcarbodiimide
  • NHS N-hydroxysuccinimide
  • flow cells 1 and 2 were blocked with 1 M ethanolamine pH 8.5 for 10 minutes.
  • Flow cell 2 has a 2024 or 2305 response unit attached to the sensor chip surface.
  • 50 mM NaCl and 1 M NaCl 10 were flown into the flow cells 1 and 2 five times, and then 10 1 of angiotensin ⁇ -pyotin (0.1 ⁇ ) was flown into the flow cell 2 in which the 2024 response unit was bonded to the sensor chip surface.
  • 3 ⁇ l of Lewis-X-sp-Piotin (0.1 ⁇ ) was flowed into flow cell 2 in which the 2305 response unit was connected to the sensor chip surface.
  • Biacore was analyzed using a PP6T buffer having a composition of 50 mM potassium phosphate, 150 mM NaCl, pH 6.0, 0.1% Tween 20 at a flow rate of 201 / min.
  • the samples obtained in [35] or [37] were injected with KINJECT. Regeneration was performed with Glycine 1.5 at 201, 50 mM NaCl, 1 M NaCl 101.
  • the response curve was calculated by combining the flow cell 2 and the flow cell 1 with the bow I to calculate the combined response unit.
  • FIG. 14 shows a selection using angiotensin II as an antigen.
  • MI3-55, MI3-42, MI3-28, MI3-41 This is a bar graph showing KD calculated by translating MI3-34, MI3-5, MI3-15, and MI3-26 using [34] and then analyzing with Biacore. It showed very high affinity values of 0.4-1.5 nM, demonstrating that the single chain antibodies selected by the method of the present invention have very high affinity.
  • FIG. 15 shows that in the selection using Lewis X as an antigen, MK1-1, MK1-24, MK2-19, and MK1-17 among the sequences converging to the sequences shown in FIG. 11 and FIG.
  • This is a bar graph showing the KD calculated by translating and analyzing with Biacore. It showed a value of 20-43 nM, indicating that the single-chain antibody selected by the method of the present invention was a high affinity antibody.
  • Fig. 16 shows that after translating MI3-55 at [34], processing it at 4 ° C, 60 ° C, or 99 ° C for 5 minutes, and then performing the processing of [35], the Biacore of [39] The results of the analysis are shown in a bar graph.
  • Blocking one (Nakarai Tester Co., Ltd.): The blocking agent diluted with Milli Q (1: 4) was caloried, and blocking was performed overnight at 4 ° C. or 1 to several hours at 25 ° C. Next, the plate was washed four times with PBS (Nacalai Tester Co., Ltd.) 200 1 and the sample obtained in [35] or [37] was added to 100 1 plate. At this time, in order to examine antagonistic inhibition, a mixture of angiotensin II or Free Lewis X and a single-chain antibody sample in advance was added.
  • PBS Nacalai Tester Co., Ltd.
  • Multiskan JX (Dainippon Pharmaceutical Co., Ltd.) 450 nm and 630 nm were measured with a 96-well microplate reader, and the value of 630 nm was subtracted from the value of 450 nm.
  • Figure 17 shows that MI3-55 was translated at [34] and then treated at 4 ° C or 60 ° C or 99 ° C for 5 minutes.
  • the undiluted solution treated at 99 ° C for 5 minutes had a 0.5 dilution to 99% of 21% of the value treated at 4 ° C for 5 minutes.
  • C, treated for 5 minutes had a 44% value compared to that treated at 4 ° C, 5 minutes, but the same value was obtained by diluting the treated at 4 ° C, 5 minutes to 0.125, respectively.
  • MI3-55 treated at 99 ° C. for 5 minutes and treated in [35] has a binding activity of about 12.5% or more, and the single-chain antibody selected by the method of the present invention is extremely low. It was shown to be heat resistant.
  • the present invention is a technique for applying protein functional maturation to an evolutionary molecular engineering technique (IW method) to rapidly and inexpensively select a protein in a test tube.
  • a desired protein can be obtained by constructing a protein cDNA library, converting to an assigning molecule (IW) library, selecting, collecting genes, and repeating this cycle several times.
  • IW assigning molecule
  • a library obtained by adding a mutation (point mutation, DNA shuffling) to a known protein gene it is possible to select a protein with higher functionality, higher stability, and higher expression. is there.
  • Preliminary heat treatment before binding the library to the antigen denatures the unstable protein group to ensure that the three-dimensional structure is maintained stably or that the protein is quickly unwound. Only high protein groups can be selected.
  • the obtained protein can be expressed in a cell-free translation system in the presence of a reducing agent such as DTT, or expressed in large quantities in the cytoplasm of Escherichia coli in a reductive environment, and is stable to reducing agents. is there.

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Abstract

L'invention concerne un procédé de sélection rapide et efficace d'une protéine à haute fonctionnalité ou d'un acide nucléique codant la protéine, par sélection d'une protéine interagissant avec une molécule cible ou d'un acide nucléique codant la protéine. Le procédé consiste à: a) créer une bibliothèque d'ADN codant la protéine; b) transcrire les ADN dans la bibliothèque créée en a), attacher un bras contenant puromycine à l'extrémité 3' des ARN transcrits, puis créer une bibliothèque de molécules affectées au génotype et au phénotype dans un système de traduction exempt de cellules; c) chauffer la bibliothèque de molécules affectées; d) lier chaque molécule affectée à la molécule cible, puis laver à fond, éluer et amplifier le fragment d'acide nucléique par RT-PCR ou PCR.
PCT/JP2004/015290 2003-10-15 2004-10-15 Procede de selection rapide et efficace de proteines a haute fonctionnalite, proteines a haute fonctionnalite obtenues par ce procede, et procede de production et d'utilisation desdites proteines WO2005035751A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2024039920A1 (fr) * 2022-08-15 2024-02-22 Mbrace Therapeutics, Inc. Procédés acellulaires de production d'anticorps dirigés contre des cibles intracellulaires

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WO1998016636A1 (fr) * 1996-10-17 1998-04-23 Mitsubishi Chemical Corporation Molecule permettant d'homologuer un genotype et un phenotype, et utilisation de celle-ci

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WO1998016636A1 (fr) * 1996-10-17 1998-04-23 Mitsubishi Chemical Corporation Molecule permettant d'homologuer un genotype et un phenotype, et utilisation de celle-ci

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MAO S. ET AL.: "Phage-display library selection of high-affinity human single-chain antibodies to tumor-associated carbohydrate antigens sialyl Lewisx and Lewisx", PROC. NATL. ACAD. SCI., vol. 96, no. 12, 8 June 1999 (1999-06-08), USA, pages 6953 - 6958, XP002983603 *
MIYAMOTO-SATO E. ET AL.: "Highly stable and efficient mRNA templates for mRNA-protein fusons and C-terminally labeled proteins", NUCLEIC ACIDS RES., vol. 31, no. 15, August 2003 (2003-08-01), XP002983602 *
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RYABOVA L. A. ET AL.: "Functional antibody production using cell-free translation: effects of protein disulfide isomerase and chaperones", NATURE BIOTECHNOLOGY, vol. 15, no. 1, January 1997 (1997-01-01), pages 79 - 84, XP002041753 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024039920A1 (fr) * 2022-08-15 2024-02-22 Mbrace Therapeutics, Inc. Procédés acellulaires de production d'anticorps dirigés contre des cibles intracellulaires

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