WO2003106675A1 - Matrices de traduction et leur bibliotheque, proteines synthetisees a partir de ces matrices et bibliotheque de proteines, leurs constituants, procede de production desdites matrices et procede d'utilisation desdites matrices - Google Patents

Matrices de traduction et leur bibliotheque, proteines synthetisees a partir de ces matrices et bibliotheque de proteines, leurs constituants, procede de production desdites matrices et procede d'utilisation desdites matrices Download PDF

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WO2003106675A1
WO2003106675A1 PCT/JP2003/007508 JP0307508W WO03106675A1 WO 2003106675 A1 WO2003106675 A1 WO 2003106675A1 JP 0307508 W JP0307508 W JP 0307508W WO 03106675 A1 WO03106675 A1 WO 03106675A1
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protein
region
translation
molecule
sequence
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PCT/JP2003/007508
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English (en)
Japanese (ja)
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宮本　悦子
柳川　弘志
高嶋　秀昭
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学校法人 慶應義塾
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Priority to AU2003242344A priority Critical patent/AU2003242344A1/en
Priority to JP2004513488A priority patent/JP4747292B2/ja
Publication of WO2003106675A1 publication Critical patent/WO2003106675A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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 translation template and a library thereof, a protein synthesized therefrom, a library of proteins, elements constituting the same, and methods for producing and using the same.
  • the base sequences of the genomes of various organisms are being decoded.
  • the second act of post-sequence research involves analyzing the meaning of the decoded genomic information, that is, analyzing the structure and function of genes and proteins ⁇ Saegusa A. Japan boosts proteomics and cell biology. .. Nature 401, 6751 (1999), Dalton R, Abbott A. Can researchers find recipe for proteins and chips? Nature 402, 6763 (1999)), and analysis of protein-protein and nucleic acid-protein interactions. (Etsuko Takamoto, Hiroshi Yanagawa (2000) Series Genome Science of Post-sequence 3: Proteomics, pp.
  • the 3 'UTR is generally used to improve mRNA stability and translation efficiency (Sachs AB, Samow P, and Hentzw MW Starting at the Beginning, Middle, and Ena; Translation Initiation in Eukaryotes. (1997) Cell 89, 831-838) ⁇ Methods for substitution and modification of the chemical structure of mRNA (Ueda T, Tohda H, Chikazumi N, Use of Eckstein F, Watanabe K., Cell-free translation system usine phosphorothioate-containing mRNA. Nucleic Acids Symp Ser. 1991; (25): 151-2.) Has been devised.
  • protein analysis by cell-free translation system plays an important role in various analysis tools that have been developed for post-genome functional analysis research. The main ones are listed below. In all cases, large-scale expression of proteins is essential.
  • Methods for detecting molecular interactions include surface plasmon resonance, fluorescence resonance energy transfer, fluorescence depolarization, evanescent field imaging, fluorescence correlation spectroscopy, fluorescence imaging, solid-phase enzyme immunoassay, etc. It has been known.
  • Fluorescence Correlation Spectroscopy FCS
  • FCS Fluorescence Correlation Spectroscopy
  • EV0TEC is developing a device aimed at Ultra HTS, which screens more than 100,000 samples per day.
  • FCCS fluorescence cross-correlation spectroscopy
  • a protein interaction detection system it is necessary to modify a protein with a probe such as a fluorescent dye for immobilization.
  • the present inventors have previously proposed a method of modifying the C-terminus of a protein in a translation system using a nucleic acid derivative such as puromycin (Japanese Patent Laid-Open No. 11-322781, Japanese Patent Laid-Open No. 2000-139468). This method has advantages over the conventional chemical modification method and fluorescent protein fusion method in that the function of the protein is less likely to be impaired.
  • 3 'UTR is generally used to improve mRNA stability and translation efficiency.Sachs AB, Sarnow P., and Hentzw MW Starting at the Beginning, Middle, and Ena; Translation Initiation in Eukaryotes. (1997) Cell 89, 831-838), 3, UTRs are hundreds of bases long and cannot be easily primed by PCR. Therefore, in order to use a cell-free translation system with great effort, we decided to incorporate it into a vector with a 3 'UTR and use it (Madin K, Sawasaki T, Ogasawara III, Endo III. A highly efficient and robust cell-free protein synthesis).
  • An object of the present invention is to provide a translation template which can be prepared stably, stably and efficiently perform protein synthesis in order to solve the above problems.
  • This translation template high-throughput analysis of post-genomic structure and function can be performed.
  • C-terminal labeled proteins (Labeled protein and its oroducing methoa, labeling compound to be used in the method for analyzing function of genes, 2001, USPatent 6,228,994, H. Yanagawa, N. Nemoto, E.
  • Miyamoto protein interaction analysis, and assigning molecules (Molecule that homologizes genotype and phenotype ana utilization there, 1998, PCT / JP97 / 03766 (W098 / 16636) H. Yanagawa, N. Nemoto, E. Miyamoto, Y. Fusimi) can further improve the ability to analyze genome functions and apply it to evolutionary molecular engineering.
  • the first invention of the present invention is a translation template having a high translation efficiency in which a part of a PEG spacer is ligated to the 3 ′ end of a coding region containing information to be translated into a protein, and a method for synthesizing the PEG.
  • the present invention is characterized in that it can have a functional modifier on a part of the spacer, and provides a protein synthesized by using the template and a library thereof.
  • the second invention relates to a C-terminal labeled protein synthesized using the translation template of the first invention and a method for synthesizing the same.
  • a special sequence (3 XA sequence), characterized in that the C-terminal modifier can be provided with a functional modifier, and that the C-terminal modified protein synthesized with the template It provides the riparian.
  • the third invention relates to a translation template synthesized using the translation template of the first invention, a C-terminal modified protein (corresponding molecule) or a C-terminal modified protein with a PEG spacer part, and This method is characterized in that a special sequence (A sequence) is used at the 3 'end of the coding part of the translation template, and a functional modifier is added to a part of the PEG spacer. And a C-terminal-modified protein synthesized by the template and a library thereof.
  • the first invention of the present invention relates to a translation template comprising a code part having information to be translated into a protein and a part of a PEG spacer.
  • the coding part is information to be translated into a protein, and may have any sequence.
  • the coding part has an receptor region (A sequence) at the 3 ′ end region of the coding part, or the 3 ′ terminal region of the coding part. It has a receptor region (A sequence) and a translation-enhancing sequence (X sequence) 5 ′ upstream of the A sequence.
  • a sequence of the coding part it contains a short poly A sequence.
  • Some PEG spacers are polyethylene glycol Region, a donor region for linking to the code part, and a CCA region at the 3 'end.
  • a part of the PEG spacer may be a donor region alone or a CCA region alone, but preferably has a configuration including a PEG region containing polyethylene glycol as a main component.
  • the CCA region may be characterized by having no function of binding to a protein translated by the translation template by a peptide transfer reaction.
  • the molecular weight of the polyethylene glycol in the PEG region may be characterized as being 400 or more.
  • the function imparting unit (F1 and / or F2) may be characterized by immobilizing or fluorescently labeling the translation template and / or the protein translated from the translation template.
  • Piotin or the like can be considered as the immobilizing substance
  • fluorescein, Cy5, or rhodamine green (RhG) can be considered as the fluorescent substance. It covers these coding molecules, translation templates, and their libraries, as well as proteins translated on ribosomes and their libraries.
  • the translation template may consist of only the code part (code molecule).
  • the second invention of the present invention relates to a protein translated by the translation template of the first invention and labeled with a C-terminal by a modifying agent.
  • the translation template consists of a coding part having information to be translated into a protein and a part of a PEG spacer mainly composed of polyethylene glycol.
  • the coding part has an A sequence and an X sequence, and includes a short poly A sequence as the A sequence. It is characterized in that it has a C (or G) marauding C (or G) sequence as the X sequence, for example, an Xhol sequence.
  • Part of the PEG spacer is characterized in that the polyethylene glycol has a molecular weight of 400 or more in the PEG region containing polyethylene glycol as a main component, and at least one in the donor region and / or the CCA region. It may be characterized in that it includes three function giving units (F).
  • the CCA region may be characterized in that it has no function of binding to a protein translated by the translation template by a transpeptidation reaction.
  • the functional modifier (F1 and / or F2) may be characterized by immobilizing or fluorescently labeling the translation template and / or a protein translated from the translation template. Piotin and the like are considered as immobilizing substances, and fluorescein, Cy5, or Rhodamine Green (RhG) can be considered.
  • the modifying agent is composed of a modifying portion for labeling the C-terminus of the protein, a peptide acceptor portion containing puromycin, and a nucleotide linker connecting them. Further, the modifying agent may be characterized in that the modifying portion contains a functional modifying substance (F3).
  • the F3 is characterized in that the protein translated by the translation template is immobilized or fluorescently labeled. Biotin and the like can be considered as immobilizing substances, and fluorescein, Cy5, or rhodamine green (RhG) can be considered as fluorescent substances.
  • the present invention relates to a protein and a library of proteins characterized in that the coding part, the translation template, and the library thereof are synthesized by being translated on a ribosome in the presence of a modifying agent.
  • the third invention of the present invention relates to a protein (two associating molecules) C-terminal modified by a translation template.
  • a translation template is composed of a coding part having information to be translated into a protein and a part of a PEG controller. It has an A sequence at the 3 'end of the coding region, and the A sequence includes a short poly A sequence.
  • Part of the PEG spacer is characterized in that polyethylene glycol has a molecular weight of 400 or more in the PEG region containing polyethylene glycol as a main component, and at least one in the donor region and / or the CCA region. And two modifiers (F1 and / or F2).
  • the CCA region is characterized in that it has a function of binding to a protein translated by the translation template by a peptide transfer reaction, and typically has pieuromycin in the CCA region.
  • the modifying substance (F1 and / or F2) may be characterized in that the translation template and / or the protein translated from the translation template is immobilized or fluorescently labeled. Biotin and the like can be considered as immobilizing substances, and fluorescein, Cy5, or rhodamine green (RhG) can be considered as fluorescent substances.
  • the present invention relates to a protein (two-mapping molecule) and a library of proteins (two-mapping molecule) synthesized by translating the coding part, the translation template, and the library on the ribosome.
  • a protein two-mapping molecule
  • a library of proteins two-mapping molecule synthesized by translating the coding part, the translation template, and the library on the ribosome.
  • it is possible to synthesize proteins in cell-free translation systems and cells using coding molecules, translation templates, and their libraries, and realize protein structure and function analysis. it can.
  • mapped molecules can be used to create functional substances based on molecular evolution engineering and to analyze genomic functions ( Figure 4).
  • protein-substance interaction analysis characterized by fluorescently labeling and / or immobilizing the translation template and its library and / or the protein and its library is also possible ( ( Figure 5).
  • various types of affinity screening, microarrays and protein groups are examples of immobilization
  • FCCS analysis is an example of fluorescent labeling ( Figure 5). The above analysis can be used in combination with in vitro cotranslation and cotranslation screening methods.
  • the present invention more specifically provides the following.
  • a coding molecule comprising:
  • the coding molecule according to 1, wherein the except region comprises a poly.A sequence having a length of 2 to 10 bases.
  • a library of the coding molecules according to any one of 1 to 4 ′.
  • a PEG spacer having a coding part having information to be translated into a protein and at least a PEG spacer having a donor region for linking to the coding part, wherein the coding part is described in any one of 1 to 4. Translation template derived from the coding molecule.
  • a PEG spacer characterized in that a part of the spacer has a donor region for linking to the coding part, a PEG region containing polyethylene glycol as a main component, and a CCA region at the 3 ′ end. 6. Translation template according to 6 derived from molecule.
  • the CCA region comprises a protein translated by the translation template and a peptide.
  • a library of proteins which is synthesized by translating a library of translation templates described in 17.15 on ribosomes.
  • a protein wherein the translation template according to any one of 8.6 to 14 comprises a modifying portion, a peptide acceptor portion containing a pure mouth mycin, and a nucleotide linker connecting them.
  • the translation template described in 23.15 labeled the C-terminus of the protein, including the modified part, the peptide acceptor containing Beauomycin, and the linker of the nucleotide linking them.
  • a library of proteins which is synthesized by being translated on a ribosome in the presence of a modifying agent.
  • coding part 30 includes a coding part having information to be translated into protein and at least a PEG spacer part having a donor region for linking to the coding part, and the coding part is derived from the coding molecule described in 28. Translation template to do.
  • a part of the donor has a donor region for connection to the code part, a PEG region mainly composed of polyethylene glycol, and a PEG region characterized by having a CCA region at the end.
  • the protein according to any one of 16, 16, 18 to 23, 35, and 37, or the protein according to any one of 17, 23 to 27, 36, and 38 A method for analyzing the interaction between a protein and a substance, comprising analyzing the interaction by causing a substance to interact with a library of proteins.
  • Interaction analysis can be performed 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.
  • 40 The method for analyzing interaction between a protein and a substance according to 40.
  • the interaction between the protein and the substance is detected by amplifying the base sequence of the coding region linked to the C-terminal of the protein.
  • FIG. 1 shows the structure of the translation template (A) of the present invention and the constituent molecules of the coding molecule (B) and the spanner molecule (C).
  • the translation template consists of a coding part derived from a code molecule and a part of a spacer derived from a spacer molecule.
  • F1 and F2 are fluorescent dyes.
  • FIG. 2 shows the configurations of the C-terminal modified protein (C-terminal labeled protein) (A), the translation template (B) of the present invention, and the modifying agent (C).
  • FIG. 3 shows the configurations of the C-terminal modified protein (assigning molecule) (A), the translation template (B) of the present invention, and the modifying agent (C).
  • FIG. 4 is an explanatory diagram of the primary screening of the interaction analysis of substances and proteins by the assigning molecule of the present invention.
  • Assignment molecules can be applied to the creation of substances that have evolved progressively from random libraries, etc., and have acquired desired functions, as evolutionary molecular engineering.
  • genome function analysis a group of gene sequences having an interaction with a desired substance / protein can be comprehensively analyzed from a cDNA library.
  • a cell-free cotranslation screening method can also be used.
  • FIG. 3 shows explanatory diagrams of primary screening and secondary screening of action analysis.
  • C-terminal modified protein synthesized from the translation template of the present invention protein modified at the C-terminal with a modifying agent, protein modified at the C-terminal with a translation template (assigning molecule), and protein modified at the C-terminal with PEG is used.
  • the screened protein in Fig. 4 can be used as a primary screening to further analyze the details of the interaction with the substance-protein using FCCS, microarray, etc. is there.
  • Figure 2 shows a comparison of the translation amount when the translation template has no PEG spacer part and when the translation template has a different PEG spacer part.
  • FIG. 8 shows the translation amount and stability of the translation template.
  • FIG. 9 shows a comparison of the amount of translation when the translation template of the present invention has a different part of PEG (including the results of electrophoresis (photograph)).
  • mapping molecule Formation of the mapping molecule due to the difference in the amount of translation template added when the coding molecule (XA sequence, A sequence) having the C-jun gene sequence and dCdCT (Flu) PEG4000 dCdCPuro-Boc are compared. efficiency.
  • FIGS. 10 to 14 show one PEG spacer molecule used in the present invention and a synthesis scheme thereof.
  • FIGS. 15 to 18 show the modifying agents used in the present invention and their synthesis schemes.
  • Figure 19 outlines the structures of the assigning molecule, the spacer molecule and the coding molecule.
  • FIG. 20 shows a detailed configuration of an example of the spacer molecule.
  • D Donor area
  • X2 and XI Functional unit
  • PEG PEG area
  • A Peptide accept area.
  • Bio Piotin
  • F1 Fluorescent dye.
  • FIG. 21 shows a detailed configuration of an example of a coding molecule.
  • FIG. 22 is a diagram showing the structures of the C-terminal modified protein (A), the modifying agent (B), and the translation template (C).
  • the assigning molecule means a molecule that associates a phenotype with a genotype.
  • the assigning molecule is a combination of a gene type molecule containing a nucleic acid having a nucleotide sequence reflecting a genotype and a phenotype molecule containing a protein involved in phenotypic expression.
  • a genotype molecule is formed by combining a coding molecule having a base sequence reflecting a genotype in such a form that the base sequence can be translated, and a part of a spacer.
  • the part derived from the phenotype molecule, the part derived from the spacer molecule, and the part derived from the coding molecule are called a decoding part, a part of the spacer, and a coding part, respectively.
  • a portion derived from a spacer molecule and a portion derived from a coding molecule are referred to as a spacer portion and a coding portion, respectively.
  • Figure 19 shows the rough structure of an example of the assigning molecule, one spacer molecule, and one example of the coding molecule.
  • This assigning molecule is composed of a base sequence (called a coding part) that reflects the phenotypic code and a primer (called a part of the spacer) containing puromycin.
  • This assigning molecule is linked to a coding molecule by a method and a part of a peptide containing puromycin to form a genotype molecule, which is linked to a phenotype molecule on a liposome in a cell-free translation system. Has a configuration.
  • One spacer molecule is composed of a PEG region composed mainly of polyethylene glycol, a CCA region composed of at least puromycin or puromycin and one or more residues of DNA and / or RNA, and MA and / or at least one residue. Or a donor region containing A, and a function-imparting unit (X) in which at least one base of DNA and / or RNA is functionally modified.
  • the coding molecule includes a DNA and / or RNA poly-A sequence consisting of a part of the sequence of the decoding region and a terminal region, and a transcription promoter and a translation enhancer consisting of DNA and / or RNA.
  • the spacer molecule consists of a donor region that can bind to the 3 'end of nucleic acid, a PEG region containing polyethylene glycol as a main component, which is bound to a donor region, and a peptide that is bound to the PEG region by a transpeptidation reaction. And a peptide receptor region containing a group capable of binding.
  • the donor region that can bind to the 3 'end of a nucleic acid usually consists of one or more nucleotides.
  • the number of nucleotides is usually 1-15, preferably 1-2.
  • the sequence at the 5 'end of the nucleic acid donor region determines the ligation efficiency.
  • at least one residue dC is required. (Doxycytidylic acid) or two residues of dC dC (dideoxycytidylic acid) are preferred.
  • the type of base is preferably in the order of C> U or T> G> A.
  • the PEG region is mainly composed of polyethylene glycol.
  • the term “main component” means that the total number of nucleotides contained in the PEG region is 20 bp or less, or the average molecular weight of polyethylene glycol is 400 or more. Preferably, it means that the total number of nucleotides is 10 bp or less, or the average molecular weight of polyethylene glycol is 1000 or more.
  • the average molecular weight of polyethylene glycol in the PEG region is usually from 400 to 30,000, preferably from 1,000 to 10,000, more preferably from 2,000 to 8,000.
  • the molecular weight of the polyethylene glycol is lower than about 400, when the genotype molecule containing a part of the spacer derived from this one spacer is mapped and translated, the post-processing of the mapped translation is performed.
  • the peptide region is not particularly limited as long as it can bind to the C-terminus of the peptide.
  • puromycin 3, -N-aminoacylpuromycin aminonucleoside (3, -N-Aminoacy lpurorayc in aiinonucleoside)
  • PANS-Gly whose amino acid portion is glycine, PANS-Val of palin, PANS-Ala of alanine, and PANS-all amino acids corresponding to all amino acids can be used.
  • 3'-N-aminoacyl adenosine aminonucleoside 3, -Aminoacyladenosine aminonucleosiae, AAN
  • AANS-Gly of glycine, AANS-Val of palin, AANS-Ala of alanine, and other AANS-all amino acids corresponding to all amino acids can be used.
  • nucleosides or those in which a nucleoside and an amino acid are ester-linked can also be used.
  • all of the nucleosides or substances having a chemical structure skeleton similar to nucleosides and substances having a chemical structure skeleton similar to amino acids or amino acids can be used as long as they can be chemically bonded.
  • the peptide peptide region is preferably composed of puromycin or a derivative thereof, or puromycin or a derivative thereof and one or two residues of deoxyribonucleotides or ribonucleotides.
  • the derivative means an derivative capable of binding to the C-terminus of the peptide in a protein translation system.
  • Puromycin derivatives are not limited to those having a complete puromycin structure, but also include those in which a portion of the puromycin structure is missing. Specific examples of puromycin derivatives include PANS-amino acids, AANS-amino acids, and the like.
  • the peptide acceptor region may be composed of pure mouth mycin alone, but preferably has a nucleotide sequence comprising one or more residues of DNA and / or RNA on the 5 ′ side.
  • the sequence may be dC-puromycin, rC-puromycin, etc., more preferably dCdC-puromycin, rCrC-puromycin, rCdC-puromycin, dCrC-puromycin, etc. 3.
  • CCA sequences that mimic the ends Philipps, GR (1969) Nature 223, 374-377) are suitable.
  • the type of base is preferably C> U or T>G> A.
  • the spacer molecule preferably contains at least one function-imparting unit between the donor region and the PEG region.
  • the function-imparting unit is preferably one in which at least one residue of deoxyribonucleotide or ribonucleotide base is functionally modified.
  • examples of the function modifying substance include substances for immobilization and fluorescent labeling. As a specific example, it is possible to introduce the fluorescent substance shown in FIG. 20, biotin, or various separation tags such as His-tag.
  • FIG. 20 shows a detailed configuration of an example of a spacer molecule.
  • a spacer molecule is composed of a PEG region composed mainly of polyethylene glycol, a CCA region composed of puromycin or puromycin and at least one residue of DNA and / or RNA, and at least one residue of DNA and / or RNA. Or a donor region containing RNA, and a function-imparting unit (X) in which at least one base of DNA and / or RNA is functionally modified.
  • a fluorescent substance T (F1) and a biotin T (Bio) are used as the function imparting unit (X).
  • the coding molecule is a 5 'untranslated region containing a transcription promoter and a translation enhancer, a 3' side of the 5 'untranslated region, a protein-encoding 0RF region, and a 3' side of the 0RF region.
  • the coding molecule may be DNA or RNA, and in the case of RNA, the terminal may or may not have a Cap structure.
  • the coding molecule can be integrated into any vector or plasmid.
  • the 3 'terminal region contains, for example, an Xhol sequence and a poly A sequence downstream thereof.
  • the poly A sequence in the 3 'terminal region is important, and the poly A sequence has at least 2 dA and / or rA residues.
  • a single poly A continuous chain preferably a poly A continuous chain having 3 or more residues, more preferably 6 or more, and still more preferably 8 or more residues.
  • Factors affecting the translation efficiency of the coding molecule include a combination of a 5 'UTR consisting of a transcription promoter and a translation enhancer and a 3' end region containing a poly A sequence. You. The effect of the poly A sequence in the 3 'terminal region is usually exerted with 10 residues or less.
  • T7 / T3 or SP6 can be used, and there is no particular limitation. SP6 is preferred, and SP6 is particularly preferred when an omega sequence or a sequence containing a part of an omega sequence is used as the enhancer sequence for translation.
  • the translation enhancer is preferably a part of an omega sequence, and the omega sequence may be a TMV omega sequence (Gallie DR, Walbot V. (1992) Nucleic Acids Res., Vol. 20, 4631-4638).
  • the one containing a part of (029) is preferable.
  • the combination of the Xhol sequence and the polyA sequence is important. Also important is the combination of the polyA sequence with the affinity tag attached downstream of the 0RF region, ie, upstream of the Xhol sequence.
  • the affinity sequence is not limited as long as it is a sequence for using any means capable of detecting a protein such as an antigen-antibody reaction. Preferably, it is a Flag-tag sequence which is a tag for affinity separation and analysis by an antigen-antibody reaction.
  • the translation efficiency increases when the Xho I sequence is added to the affinity tag such as Flag-tag and when the poly A sequence is further added thereto.
  • the configuration effective for the translation efficiency described above is also effective for the association efficiency.
  • the 0RF region may be any sequence consisting of DNA and / or RNA. Gene sequences, exon sequences, intron sequences, random sequences, or any natural or artificial sequences are possible, and there are no sequence restrictions.
  • each length is about 60 bp in 5, UTR, 3.
  • Approximately 40 bp in the terminal region enough to be incorporated as an adapter region into PCR primers. For this reason, it is easy to generate a code molecule with 5, UTR and 3, terminal regions from any vector plasmid cDNA library by PCR.
  • translation may be made beyond the 0RF region. That is, a termination codon may not be present at the end of the 0RF region.
  • Figure 21 shows the detailed structure of an example of a coding molecule.
  • the coding molecule consists of a 3 'terminal region, a 5' UTR containing a transcription promoter composed of DNA and / or RNA and a translation enhancer, and sequence information of a decoding region, that is, a 0RF region encoding a phenotypic protein.
  • DNA and / or R It contains an affinity tag sequence consisting of NA, an Xhol sequence, and a poly A sequence, and uses a Flag-tag sequence. 5.
  • a sequence containing SP6 of the transcription promoter and 029 which is a part of the omega sequence of the translation enhancer is used as the UTR.
  • the genotype molecule (translation template) is composed of a 5 'untranslated region containing a transcription promoter and a translation enhancer, a 0RF region encoding a protein bound to the 3' side of the 5 'untranslated region, and a 3' region of the 0RF region.
  • the 3 'end of a coding molecule which is a nucleic acid containing a 3' end region containing a poly A sequence, and which is linked to the 'side, is linked to the donor region of one spacer molecule.
  • the code molecules that make up the genotype molecule are as described for the coding molecule. However, it is preferable to have an expression enhancing sequence if necessary.
  • a genotype molecule can be produced by linking the 3 'end of the above-mentioned coding molecule to the donor region of a spacer molecule by a usual ligase reaction.
  • the reaction conditions are generally 4 to 48 hours at 4 to 25 ° C, and polyethylene glycol having the same molecular weight as the polyethylene glycol in the PEG region of a spacer molecule containing the PEG region is used. When added to the reaction system, it can be reduced to 0.5 to 4 hours at 15 ° C.
  • spacer molecules and coding molecules has a significant effect on ligation efficiency.
  • the 3'-terminal region of the coding region corresponding to the receptor there is at least 2 or more, preferably 3 or more, more preferably 6 to 8 or more DNA and / or RNA poly A sequence.
  • a 5 'UTR translation enhancer a partial sequence of an omega sequence (029; FIG. 21) is preferable, and a donor region of a spacer is at least one residue of dC (doxycytidylic acid) or 2 The residue dCdC (dideoxycytidylic acid) is preferred. This makes it possible to avoid the problems of DNA ligase by using RNA ligase, and to maintain the efficiency at 60 to 80%.
  • genotype molecule is RNA
  • the 3 'end of the coding molecule and (b) the donor region of the spacer molecule, which is composed of RNA, are the same as the polyethylene glycol constituting the PEG region in the spacer molecule. It is preferable to bind with RNA ligase in the presence of free polyethylene glycol having a molecular weight.
  • the ligation efficiency can be 80-90 regardless of the molecular weight of the part of the polyethylene glycol. %, And the separation step after the reaction can be omitted.
  • the assigning molecule is obtained by linking the above-mentioned genotype molecule to a phenotype molecule which is a protein encoded by the 0RF region in the genotype molecule in a transpeptidation reaction.
  • the assigning molecule is a phenotype that is a protein encoded by the 0RF region in the genotype molecule in a transpeptidation reaction by translating the genotype molecule on a ribosome (for example, by a cell-free translation system). By linking to a molecule.
  • the cell-free translation system is preferably of wheat germ or egret reticulocytes.
  • the translation condition may be a condition that is usually adopted. For example, a condition at 25 to 37 ° C. for 15 to 240 minutes can be mentioned.
  • mapping molecules As for the cell-free translation system, the formation of mapping molecules has been examined in E. coli (E. coli), Egret reticulocytes, and wheat germ systems, and the association molecules have been confirmed only in the Eg reticulocyte system.
  • Taga Nemoto, N, Miyamoto-Sato, E., Yanagawa, H. (1997) FEBS Lett. 414, 405; Roberts, RW, Szostak, JW (1997) Proc. Natl. Acad. Sci. USA 94,
  • the mapping molecule can be formed even in a wheat germ system.
  • heron reticulocyte system was not practical because of the lack of stability of the genotype molecule, and has been applied only to short-chain genotype molecules (Roberts, RW, Szostak, JW (1997) Natl. Acad. Sci. USA 94, 12297; Nemoto, N., Miyamoto-Sato, E., Yanagawa, H. (1997) FEBS Lett. 414, 405), part of spacer containing PEG region Are more stable in wheat germ systems and are practical for handling long chain lengths. System.
  • the C-terminal labeled protein is a protein having a modified C-terminus, and has a configuration in which a labeling agent is bonded to the C-terminus of the protein, as shown in FIG. 22A. That is, the C-terminal labeled protein is composed of the protein and a labeling agent.
  • Protein that constitutes a C-terminally labeled protein means a protein used as an analysis target for an interaction whose function is known or unknown.
  • the C-terminally labeled protein of the present invention can be used for measuring the presence or absence of an interaction between this protein and a target molecule described below.
  • This protein may be a natural protein or a variant thereof, and an artificial protein or a variant thereof.
  • Natural proteins also include libraries of diverse proteins transcribed and translated from cDNA libraries derived from various organs, tissues or cells of organisms.
  • the artificial protein is a sequence obtained by combining all or a partial sequence of the natural protein, or a sequence containing a random amino acid sequence.
  • the protein constituting the C-terminal labeled protein is preferably a full-length protein.
  • full-length protein refers to a protein in which the C-terminus is completely translated, that is, a protein obtained by translating the codon up to one stop before the stop codon of the nucleotide sequence encoding the protein. means.
  • the N-terminus of the full-length protein may have undergone some processing such as cleavage of a signal peptide.
  • the protein constituting the C-terminal labeled protein may be a protein fused with an affinity tag.
  • affinity tags include polyhistidine peptide peptide peptides, glutathione-S-transferase, protein A, maltose binding protein, calmodulin binding peptide and the like.
  • a C-terminal labeled protein can be produced by expressing a translation template in a translation system in the presence of a labeling agent, performing protein synthesis, and purifying the synthesized protein.
  • a labeling agent examples of the labeling agent, the translation template, and the production will be described, but the present invention is not limited thereto.
  • the labeling agent reacts with the transpeptidase in the protein translation system.
  • a peptide acceptor having a group (including a residue) capable of binding to a protein by a transpeptidation reaction on a ribosome has a structure in which the moiety is linked to a modified portion via a nucleotide linker.
  • the modifying substance included in the modifying portion include fluorescent and non-fluorescent modifying substances.
  • the fluorescent substance 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).
  • the non-fluorescent substance may be any compound such as a coenzyme such as biotin, a protein, a peptide, a saccharide, a lipid, a dye, polyethylene glycol, etc., as long as it can be any marker.
  • the modified moiety has a fluorescent group, a group that binds to a protein (for example, a piotinyl group or an iminobiotinyl group), or both.
  • a piotinyl group or an iminobiotinyl group because the efficiency of modification with the C-terminal labeling agent of the present invention increases.
  • a part of peptide peptide is a protein translation system and has a group capable of binding to a protein by a transpeptidation reaction, and preferably has a residue of pure mouth mycin or a derivative thereof.
  • Puromycin has a similar structure 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 in the present invention may be any substance having a structure similar to puromycin and capable of binding to the C-terminus of a protein. Specific examples include 3, -N-aminoacylbiuromycin amino nucleoside, 3, -N-aminoacyl adenosine amino nucleoside, and the like.
  • Nucleotide linkers that connect between the modified moiety and a portion of the peptide acceptor are, specifically, nucleic acids or nucleic acid derivatives in which one or more ribonucleotides or deoxyribonucleotides are linked, and particularly preferred examples.
  • cytosine Examples include compounds in which one or more ribonucleotides containing a base (-rC-) or deoxyribonucleotides (-dC-) are linked.
  • any substance can be used as long as it can increase the yield of the modified protein by being inserted between the modification part and a part of the peptide receptor.
  • the nucleotide linker is 2'-deoxycytidylic acid, 2, -deoxycytidyl- (3,5,)-2, -deoxycytidylic acid, ribocytidylic acid, or ribocytidyl- (3,5,5)-.
  • it is ribocytidylic acid.
  • the labeling agent can be produced by binding the above-mentioned modified moiety and a part of the peptide receptor via a desired nucleotide linker by a chemical bonding method known per se. Specifically, for example, a portion of the above peptide acceptor protected with an appropriate protecting group is bound to a solid support, and a nucleotide phosphoramidite and a nucleotide are converted into a nucleotide linker using a nucleic acid synthesizer or the like.
  • It can be prepared by sequentially binding xynucleotide phosphoramidite and a nucleotide phosphoramidite to which a fluorescent substance, a biotin or the like is bound as a modifying substance, and then performing deprotection. Depending on the type of each part or the type of bonding, they can be combined by a liquid phase synthesis method, or both can be used in combination.
  • a metal ion such as nickel
  • a chelating reagent such as nitrilotriacetic acid or iminodiacetic acid to which the metal ion can coordinate can be bound, and then the metal ion can be coordinated.
  • the translation template is a translation template that can be used when producing the modified protein of the present invention. As shown in FIG. 22C, the 3 ′ end region containing the poly A sequence and the 5 ′ It consists of the untranslated region (5 'UTR) and the encoded 0RF region of the protein.
  • the translation template may be DNA or RNA.
  • the translation template comprises a 0RF region encoding a protein, located on the 5th side of the 0RF region, a 51TR containing a transcription promoter and a translation enhancer, and located on the 3 'side of the 0RF region, It is composed of three terminal regions containing a poly A sequence (polyA).
  • a more preferred translation template is SP6 RNA as a transcription promoter for UTR. Contains the promoter sequence of the polymerase and a part of the omega sequence of tobacco mosaic virus (TMV) (029) as a translation enhancer. Further, it is preferable that the 0RF region contains an affinity tag sequence in a downstream portion thereof.
  • the affinity tag sequence is a sequence encoding the affinity tag described above, and preferably includes a His-tag (polyhistidine tag) sequence.
  • a longer polyhistidine tag is preferable because the recovery rate by the nickel chelate resin is improved.
  • the preferred range of the length of the polyhistidine tag can vary depending on the type of protein to be modified and the type of label, but is usually 8 to 12 residues.
  • upstream and downstream mean in the direction of transcription or translation.
  • the translation template when it is DNA, it may be a DNA vector or a plasmid obtained by introducing the above region into an appropriate DNA vector or brasmid. If the translation template is MA, it may or may not have a Cap structure at the end.
  • Examples of translation systems used for producing C-terminal labeled proteins include cell-free protein synthesis systems and cell expression systems.
  • specific examples of the cell-free protein synthesis system include a wheat germ extract, a heron reticulocyte extract, and an Escherichia coli S30 extract.
  • the translation template described above is added to these cell-free protein synthesis systems, the modifier is added at the same time at 1 to 100 / M, and the mixture is incubated at 25 to 37 ° C for 1 to several hours to synthesize a C-terminal modified protein. You.
  • the synthesized modified protein can be directly used for the next purification process or detection process.
  • cell expression systems include bacteria such as Escherichia coli, Bacillus subtilis, thermophiles, and yeast, cultured cells such as insect cells, mammals, and the like, as well as nematodes, Drosophila, zebrafish, and mice. Any cell can be used as long as gene transfer is possible, up to the cell.
  • the above-mentioned translation template of the present invention is introduced into these cells, and at the same time, 1 to 100 / M of the modifying agent of the present invention is introduced into the cells by electroporation, micro-injection method, or the like.
  • the modified protein is synthesized by incubating at the growth temperature for several hours.
  • the synthesized modified protein is The cells can be recovered by disruption and subjected to subsequent purification or detection processes. It can also be subjected to the detection process in cells as it is. Select a translation template that is appropriate for the translation system used.
  • any method generally used for protein purification such as affinity, chromatography such as gel filtration and ion exchange, electrophoresis, precipitation, and dialysis, can be used.
  • affinity chromatography, gel filtration, ion chromatography, electrophoresis, precipitation, dialysis, and any combination thereof are included.
  • Particularly preferred examples include modified proteins obtained by fusing an affinity tag such as polyhistidine peptide peptide, glutathione-S-transferase, protein A, maltose binding protein, and calmodulin binding peptide to an affinity resin.
  • an affinity tag such as polyhistidine peptide peptide, glutathione-S-transferase, protein A, maltose binding protein, and calmodulin binding peptide to an affinity resin.
  • the gel is applied several times to the gel filtration column.
  • the unmodified protein is completely removed by utilizing the affinity of the piotinyl group or iminopiotinyl group of the modified portion and avidin or streptavidin.
  • the translation template (A in FIG. 1) of the first invention is composed of a coding part derived from the coding molecule (B in FIG. 1) and a PEG spacer derived from one PEG spacer molecule (C in FIG. 1). Department.
  • the stability is improved and the translation efficiency can be improved by connecting (ligating) a part of the PEG spacer to the code part regardless of the arrangement of the code part.
  • the translation efficiency can be further improved depending on the structure of the code part and the type of the PEG spacer. The details are described below.
  • the coding region of the present invention (B in FIG. 1) comprises a 5 ′ terminal region, (ffiF region and 3 ′ terminal region, and may or may not have a Cap structure at the 5 ′ terminal.
  • the 3 'end region of the coding region contains a poly Ax 8 sequence or an X sequence as an A sequence.
  • an Xhol sequence or a sequence with 4 or more bases C or G
  • orchid C or G
  • One orchid
  • a configuration consisting of an A sequence, an X sequence, or a Flag tag sequence as an affinity tag sequence upstream of the XA sequence is 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 it doesn't matter.
  • the combination of XA sequences is important. Among the X sequences, the first four bases are important, and those having an SNNS sequence are preferable.
  • the upper limit of the length of the X sequence is not particularly limited as long as the effect of enhancing translation is obtained, but is usually 15 bases or less, preferably 6 bases or less.
  • the 5'-terminal region consists of a transcription promoter and a translation enhancer.
  • the transcription promoter can be T7 / T3 or SP6, etc., and is not particularly limited. However, in a wheat cell-free translation system, it is used as a translation enhancer sequence. It is preferable to use a sequence containing a part of an omega sequence or an omega sequence, and it is preferable to use SP6 as a promoter.
  • a portion (029) of the omega sequence of the translation enhancer contains a portion of the TMV omega sequence (Gallie DR, Walbot V.
  • the 0RF region of the coding part may be any sequence consisting of DNA and / or RNA. Gene sequences, exon sequences, intron sequences, random sequences, or any natural or artificial sequences are possible and there are no sequence restrictions.
  • a part of the PEG spacer of the present invention (C in FIG. 1) is composed of a CCA area, a PEG area, and a donor area.
  • the minimum required configuration is the donor region.
  • 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, more preferably from 2,000 to 6,000.
  • the CCA region can be configured with or without puromycin.
  • puromycin for puromycin, puromycin, 3, -N-aminoacylpuromycin aminonucleoside (3'-N-Aminoacylpuromycin aminonucleoside)
  • PANS-Gly of glycine PANS-Val of palin, PANS-Ala of alanine, and other PANS-all amino acids corresponding to all amino acids can be used.
  • 3'- 3, -N-aminoacyl adenosine aminonucleoside (3 "-Aiinoacyl adenosine aminonucleoside, AANS-amino acid) linked by an amide bond formed as a result of dehydration condensation of the amino group of aminoadenosine and the carboxyl group of amino acid
  • AANS-Gly of glycine, AANS-Val of palin, AANS-Ala of alanine, and other amino acids corresponding to all amino acids can be used for the amino acid portion, and nucleosides or ester bonds of nucleosides and amino acids.
  • any substance that has a chemical bond between a nucleoside or a substance having a chemical structural skeleton similar to a nucleoside and a substance having a chemical structural skeleton similar to an amino acid or an amino acid can be used.
  • the above pure mouth mycin induction Any substance lacking the ability of the amino group to bind to amino acids and the CCA region lacking puromycin may be considered.However, by including puromycin that cannot bind to proteins on the ribosome, translation efficiency can be further improved. For unknown reasons, puromycin, which is unable to bind to proteins, may stimulate ribosomes to promote overnight turnover.
  • a base sequence consisting of one or more residues of DNA and / or RNA
  • the type of base is preferably C> U or T>G> A
  • the sequence is dC-puromycin , RC-puromycin, etc., and more preferably dCdC-puromycin, rCrC-puremycin, rCdC-puremycin, dCrC-puremycin, etc .
  • CCA sequence that mimics an end Philipps GR (1969) Nature 223 , 3 7 4-377) are suitable.
  • these Pew port mycin is impossible coupled with an amino acid in some way .
  • a part of the PEG spacer of the present invention can be configured to have a modifying substance (F1 and / or F2).
  • the translation template can be used as a tag for recovery, reuse by purification, or immobilization.
  • the length is 60 bp, about 40 bp in the 3 'end region, and a length that can be designed as an adapter region in the primer of PCR. This has a new effect Was issued.
  • a translation template with high translation efficiency can be obtained.
  • the method of ligating the PEG sensor part and the code part of the present invention is not particularly limited, and any method may be used, such as a method using a general DNA ligase or a linkage by a light reaction.
  • the A sequence in the terminal region is important as the range that affects ligation efficiency in the coding region, and a mixture of dA and / or rA of at least 2 residues or more And preferably 3 or more residues, more preferably 6 to 8 residues or more.
  • the DNA and / or RNA sequences at the 5 'end of some of the donor regions of the PEG matrix determine the ligation efficiency.
  • the type of base is preferably C> U or T> G> A. Further, it is preferable to add polyethylene glycol having the same molecular weight as that of the PEG region at the time of the Liigession reaction.
  • the second invention is a protein (A in FIG. 2), which is synthesized by translation using the translation template of the first invention in the presence of the modifying agent and is C-terminal modified with the modifying agent. It consists of a template (B in Fig. 2) and a modifier (C in Fig. 2). The feature here is especially the structure of the code part of the translation template. The details are described below.
  • the PEG spacer part of the translation template of the present invention (B in FIG. 2) is similar to the first invention in that puromycin cannot be linked to an amino acid.
  • Approximately 60 bp, approximately 40 bp in the 3'-terminal region, and a length that can be designed as an adapter region for PCR primers This allows any vector And a plasmid containing a 5'-terminal region and a 3'-terminal region of the present invention can be easily prepared by PCR from a cDNA library. By ligating a part of the template, a translation template with high translation efficiency suitable for C-terminal labeling can be obtained.
  • the modifying agent of the present invention (C in FIG. 2) has a group (including a residue) capable of binding to the protein by a transpeptidation reaction in a protein translation system, that is, a peptide transfer reaction on a ribosome.
  • the peptide acceptor has a configuration in which the peptide acceptor is bound to the modifying moiety via a nucleotide linker.
  • the non-radioactive modifying substance as the modifying substance include fluorescent and non-fluorescent modifying substances.
  • the fluorescent substance include fluorescent dyes such as fluorescein series, rhodamine series, Cy3, Cy5, eosin series, and NBD series, and fluorescent proteins such as green fluorescent protein and (GFP).
  • any compound can be used as a marker, such as a coenzyme such as biotin, a protein, a peptide, a saccharide, a lipid, a pigment, and polyethylene glycol.
  • the modifying portion has a fluorescent group, a group binding to a protein, or both.
  • the peptide acceptor has a group capable of binding to protein by a transpeptidation reaction in a protein translation system, and preferably has a puromycin 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 in the present invention may be any substance as long as it has a structure similar to puromycin and can bind to the C-terminus of the protein.
  • Nucleotide linkers that connect between the modified moiety and a portion of the amino acid are specifically ribonucleotides or deoxyribonucleic acids.
  • a nucleic acid or nucleic acid derivative in which one or more nucleotides are linked, and particularly preferred is one or more ribonucleotides (-rC-) or deoxyribonucleotides (-dC-) containing cytosine bases.
  • the nucleotide linker is 2′-dexoxytidylic acid, 2, -dexoxytidyyl- (3 ′, 5 ′)-2, -dexoxytidylic acid, ribocytidylic acid, or ribocytidyl- (3 ′, 5 ′). ;)-Lipositidylic acid.
  • the modifying agent of the present invention can be produced by binding the above-mentioned modified moiety and a part of the peptide via a desired nucleotide linkage by a chemical bonding method known per se. Specifically, for example, a part of the above-mentioned amino acid protected with an appropriate protecting group is bound to a solid support, and a nucleotide linker is used as a nucleotide linker using a nucleic acid synthesizer. It can be produced by sequentially binding xynucleotide phosphoramidite, and a phosphoramidite to which a fluorescent substance or biotin is bound as a functional modifier, and then performing deprotection.
  • the third invention relates to a protein synthesized by translation using the translation template of the first invention and modified at the C-terminal with the translation template (A in FIG. 3; assigning molecule) and a translation template (B in FIG. 3). ) Is removed, and the protein has the structure of C-terminally modified protein with PEG (C in Fig. 3). The details are described below.
  • the PEG spacer part of the translation template (B in FIG. 3) is the same as the first invention except that puromycin can be linked to an amino acid.
  • the code part is also the same as that of the first invention, but in particular, it is important that the 3 'terminal region be an A sequence as a configuration suitable for association, It was confirmed that the efficiency of the association was significantly improved and the amount of free protein was drastically reduced.
  • the 5 'end region of the code part is SP6 + 029, and the end region is, for example, Flag + XhoI + A n (n28).
  • each length is about 60 bp at the 5′-end region and about 40 bp at the 3′-end region, and is a length that can be designed as one adapter region for PCR primers.
  • the Yotsute by hail loose vector or plasmid Doya cDNA library one to PCR which easily allows write code section having an end region and a 3 5-terminal region the end of the present invention, to Raigeshiyon the PEG spacer portion As a result, a translation template with high association efficiency can be obtained.
  • the protein modified with C-terminal by PEG of the present invention (FIG. 3C) can be used for detecting protein interaction without using the coding part, for example, for FCCS measurement, fluorescent leader, protein group, etc. In the case of application, it is preferable to intentionally cleave with RNase A or the like. Cleavage can eliminate the difficulty of detecting protein-protein interactions due to interference with the coding region.
  • the mapping molecule uses the Darwinian evolutionary mechanism as an evolutionary molecular engineering to repeat three unit operations of “Mutation”, “Selection” j, and “Amplification”. From a random library, etc., it is possible to apply it engineeringly by creating a material that gradually evolves and acquires the desired function (Fig. 4). Also, as an application to genomic function analysis, it is possible to comprehensively analyze a group of gene sequences interacting with desired substances and proteins from a cDNA library (Fig. 4). Furthermore, as shown in Fig. 5, it is possible to analyze the details of the interaction with substances and proteins using FCCS or microarray, etc., as shown in Fig. 5 as the secondary screening after the primary screening in Fig. 4.
  • the above analysis can also be used in combination with in vitro cotranslation and cotranslation screening methods. Also, when using the assigning molecule in the primary screening, the coding part of the A sequence is used, and in the secondary screening, when using the assigning molecule, the coding part of the A sequence and the C-terminal labeled protein are used. In some cases, the effects can be properly used by changing the code part of the XA sequence by priming.
  • the cell-free protein synthesis system examples include a wheat germ extract, a heron reticulocyte extract, and an Escherichia coli S30 extract.
  • a C-terminal modified protein is synthesized.
  • For mapping add the above translation template and add Simply retaining the temperature at 37 ° C for one to several hours allows the associating molecules to be synthesized. Both synthesized modified proteins can be directly used for the next purification or detection process, or directly into cells.
  • the cell expression system include any cells from bacteria such as Escherichia coli, Bacillus subtilis, thermophiles, and yeast to cultured cells of insect cells, mammals, etc., as well as nematodes, Drosophila, zebrafish, mice, etc. May be.
  • bacteria such as Escherichia coli, Bacillus subtilis, thermophiles, and yeast
  • yeast to cultured cells of insect cells, mammals, etc., as well as nematodes, Drosophila, zebrafish, mice, etc. May be.
  • the above-mentioned C-terminal-labeled or both modified proteins can be directly introduced, or, if the above-mentioned translation template of the present invention is introduced and the C-terminal is labeled,
  • the modified protein of the present invention is introduced into cells by electroporation, microinjection: xillon method, etc.
  • the associating molecule is synthesized by introducing the translation template of the present invention 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. It can also be subjected to the detection process in cells as it is. Select a translation template that is appropriate for the translation system used.
  • the present invention relates to a C-terminal modified protein synthesized from the translation template of the present invention (a protein modified at the C-terminus with a modifying agent (A in FIG. 2), and a protein modified at the C-terminus using the translation template (A in FIG. 3;
  • Fig. 3C protein modified with C-terminal by PEG
  • FIG. 5 a method for analyzing, characterized by using the modified protein of the present invention containing the protein
  • the modified protein of the present invention and the target molecule obtained above 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 two molecules in the signal emitted by the target molecule.
  • 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 solid-phase enzyme immunoassay. Test method Done by The details of these methods will be described below.
  • Target molecule means a molecule that interacts with the modified protein of the present invention, and specifically includes proteins, nucleic acids, sugar chains, low molecular weight compounds, and the like.
  • the protein is not particularly limited as long as it has an ability to interact with the modified protein of the present invention, and may be a full-length 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 translated from a synthesized peptide chain, a protein purified from a living body, or a cDNA library using a suitable translation system, and the purified protein can also be used as a target molecule.
  • the synthesized peptide chain may be a sugar protein having a sugar chain bonded thereto.
  • a purified protein having a known amino acid sequence or a protein translated and purified from a cDNA library or the like by an appropriate method can be preferably used.
  • the nucleic acid is not particularly limited as long as it has an ability to interact with the modified protein of the present invention, and DNA or RNA can also be used. Further, the nucleic acid may have a known nucleotide sequence or function, or may have an unknown nucleic acid. Preferably, those having a known function as a nucleic acid capable of binding to a protein and a base sequence, or those obtained by cleavage and isolation from a genomic library or the like using a restriction enzyme or the like can be used.
  • the sugar chain is not particularly limited as long as it has an ability to interact with the modified protein of the present invention, and may be a sugar chain having a known or unknown sugar sequence or function.
  • 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 an ability to interact with the modified protein of the present invention. Either one whose function is unknown or one whose ability to bind to a protein is already known can be used.
  • the “interactions” that these target molecules make with the modified protein of the present invention are generally defined as the covalent bond, hydrophobic bond, hydrogen bond, van der Waals bond, and electrostatic bond between protein and target molecule.
  • the term refers to the action of forces acting between molecules arising from at least one, but this term should be interpreted in the broadest sense and not in any sense in a limiting sense.
  • Covalent bonds include coordination bonds and dipole bonds.
  • the coupling by electrostatic force includes not only electrostatic coupling but also electric repulsion.
  • the interaction also includes a binding reaction, a synthesis reaction, and a decomposition reaction resulting from the above action.
  • interaction examples include binding and dissociation between an antigen and an antibody, binding and dissociation between a protein receptor and a ligand, binding and dissociation between an adhesion molecule and a partner molecule, and between an enzyme and a substrate. Binding and dissociation between nucleic acids and proteins that bind to it, binding and dissociation between proteins in the signal transduction system, glycosylation and dissociation between proteins and proteins, or glycoproteins and proteins Binding and dissociation between.
  • the target molecule to be used can be used after being modified with a modifying substance according to the embodiment.
  • Modifiers are usually selected from non-radioactive modifiers such as fluorescent materials.
  • Various fluorescent dyes that have free functional groups (for example, carboxyl group, hydroxyl group, amino group, etc.) and can be linked to the above target substances such as proteins and nucleic acids, such as fluorescein series, rhodamine series, Cy3, Cy5 , Eosin series, NBD series, etc.
  • the compound is a modifiable compound such as a dye, the type and size of the compound are not limited.
  • modifying substances those suitable for a method of measuring or analyzing a change in a signal generated based on the interaction between the target molecule and the modified protein of the present invention are appropriately used.
  • the above-mentioned modifying substance can be bound to the target molecule using an appropriate method known per se. Specifically, for example, when the target molecule is a protein, the method described above for modifying the C-terminus can be used. When the target molecule is a nucleic acid, it can be easily modified by a method such as performing PCR using an oligo DNA primer to which a modifying substance is previously bound by a covalent bond or the like.
  • the modified protein of the present invention or the target molecule used in the present invention may be bound to a solid phase depending on the embodiment.
  • the modifier used in the case of binding via the modifier is usually a molecule that specifically binds to a specific polypeptide (hereinafter sometimes referred to as a “ligand”). Is a specific polypeptide that binds to the ligand (hereinafter referred to as "ada Protein protein).
  • the adapter protein also includes a binding protein, a receptor protein constituting a receptor, an antibody, and the like.
  • adapter protein / ligand combinations include, for example, biotin-binding proteins / biotin such as avidin and streptavidin, maltose-binding proteins / maltose, G proteins / guanine nucleotides, and polyhistidine peptides / pike. Or metal ions such as cobalt, glutathione-S-transferase / glutathione, DNA-binding protein / DNA, antibody / antigen molecule (epitope), calmodulin / calmodulin-binding peptide, ATP-binding protein / Examples include ATP or various receptor proteins such as estradiol receptor protein / estradiol / ligand thereof.
  • adapter proteins / ligands include biotin-binding proteins such as avidin and streptavidin, maltose-binding proteins / maltose, and metal ions such as polyhistidine peptide / nickel or cobalt.
  • biotin-binding proteins such as avidin and streptavidin
  • maltose-binding proteins / maltose and metal ions such as polyhistidine peptide / nickel or cobalt.
  • metal ions such as polyhistidine peptide / nickel or cobalt.
  • Daryuthione-S-transferase / glucathione, antibody / antigen molecule (epitope), and the like, and a combination of streptavidin / pyotin is most preferred.
  • binding proteins are known per se, and the DNA encoding the protein has already been cloned.
  • the binding of the adapter protein to the solid surface can be carried out by a method known per se. Specifically, for example, tannic acid, formalin, glutaraldehyde, pyrvic aldehyde, bis-diazo A method using benzodizone halide, toluene-2,4-disocyanate, an amino group, a carboxyl group that can be converted into an active ester, or a hydroxyl group or an amino group that can be converted into a phosphoramidide can be used.
  • Measurement methods used include, for example, fluorescence correlation spectroscopy, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, fluorescence imaging analysis, surface plasmon resonance, solid-phase enzyme immunoassay, etc. Any system that can detect molecular interactions is available.
  • FCS Fluorescence Correlation Spectroscopy
  • This value varies with the number of particles present in the volume of space observed at a particular time.
  • the various parameters mentioned above can be calculated from the variation of this signal using an autocorrelation function.
  • Equipment for performing this FCS is also commercially available from Zeiss, etc., and analysis can be performed using these equipment also in this method.
  • the target molecule does not need to be modified.
  • molecules that are much smaller in molecular weight than the C-terminal modified protein whose interaction is to be investigated do not affect the Brownian motion of the C-terminal modified protein.
  • FCCS Fluorescence resonance energy transfer
  • FRET fluorescence resonance energy transfer
  • the FCCS method when compared to other detection systems such as the fluorescence depolarization method, the FCCS method has the advantages of requiring a smaller amount of sample, shorter detection time, and easier automation for HTS.
  • the FCCS method can provide very basic information such as the size and number of fluorescently labeled molecules, and may be used for general purposes such as surface plasmon resonance. The difference between the two is that the surface plasmon resonance method detects interactions in a protein immobilized state, while the FCCS method allows you to see interactions in a solution that is closer to the natural state. .
  • the FCCS method it is necessary to label the protein with a fluorescent dye instead of immobilizing the protein, but the present invention has made it possible to overcome this problem.
  • any method may be used for bringing the C-terminal modified protein into contact with the target molecule, as long as the two molecules are brought into contact with each other to an extent sufficient to interact with each other.
  • a number of simultaneous analyzes are performed, for example, by injecting a plurality of different C-terminally modified proteins into each of the measurement wells of the above-mentioned FCS measurement apparatus, and then adding a specific target molecule solution thereto.
  • a specific C-terminal modified 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 the interaction between the two molecules causes the fluorescence emitted from the modified molecule remaining on the solid-phased molecule to be converted to a commercially available fluorescence.
  • This is a method of measuring or analyzing using a fluorescence imaging analyzer.
  • the C-terminal modified protein or the target molecule needs to be immobilized by the method described above. Modification when the target molecule is immobilized Both available and not available are available. When used without being immobilized, it is necessary to be modified with the above-mentioned modifying substance.
  • the C-terminal modified protein either a protein immobilized through a modified portion or a protein immobilized in a portion other than the modified portion can be used.
  • a substrate for immobilizing a C-terminal modified protein or a target molecule As a substrate for immobilizing a C-terminal modified protein or a target molecule, a nitrocellulose membrane or a nylon membrane, which is usually used for immobilizing proteins or nucleic acids, or a plastic microplate, etc., is also used. be able to.
  • the method of contacting the modified target molecule or the C-terminal modified protein with the immobilized molecule may be any method as long as the two molecules are brought into contact to an extent sufficient for interaction.
  • a method in which a modified target molecule or a C-terminal modified protein is dissolved at a suitable concentration in a buffer commonly used in biochemistry at an appropriate concentration, and the solution is brought into contact with the surface of a solid phase is preferred.
  • a step of washing the excessively present modified target molecule or C-terminal modified protein with the same buffer or the like is preferably performed, and the target molecule or C-terminal modified protein remaining on the solid phase
  • the fluorescence signal emitted from the modified substance of the above or the mixed signal of the fluorescence emitted from the modified molecule immobilized on the solid phase and the fluorescence emitted from the modified molecule remaining on the solid phase is used. By measuring or analyzing, it is possible to identify a molecule that interacts with the immobilized molecule.
  • a method for performing a large number of analyzes at the same time includes, for example, a method in which a plurality of c-terminal modified proteins or a modified or unmodified target molecule is assigned to the solid phase surface and immobilized, or A method in which a plurality of types of C-terminal modified proteins or modified target molecules which are not immobilized on a type of C-terminal modified protein or a modified or unmodified target molecule are brought into contact is used.
  • the molecules that remain on the solid phase are obtained by dissociation due to differences in buffer concentration, etc., and are obtained by a known method. It can be identified by analyzing with.
  • Fluorescence resonance energy transfer is a method that uses two fluorescent dyes It is well known as an interaction detection method.
  • FRET means that when the fluorescence spectrum of one of the two fluorescent dyes (energy donor) and the absorption spectrum of the other (energy acceptor) overlap, if the distance between the two fluorescent dyes is small enough, A phenomenon in which the probability that the excitation energy excites a receptor increases before light emission from the donor occurs. Therefore, two proteins whose interaction is to be detected are labeled with a fluorescent dye that serves as a donor and an acceptor, respectively.If the donor is excited, the two proteins do not interact with each other.
  • FRET does not occur due to the large distance between the two molecules, and the fluorescence spectrum of the donor is observed.However, when the two proteins interact and the distance between the fluorescent dyes decreases, FRET reduces the fluorescence spectrum of the acceptor. As observed, it is possible to determine the presence or absence of protein-protein interaction based on the difference in the wavelength of the fluorescent spectrum.
  • the fluorescent dye a combination of fluorescein as a donor and rhodamine as an acceptor is often used. Recently, attempts have been made to observe FRET in cells and detect the interaction using a combination of mutants of fluorescent green protein (GFP) with different wavelengths.
  • the disadvantage of this method is that the two fluorescent dyes must be within 40-50 A of each other for FRET to occur, so depending on the size of the protein and the location of the fluorescent dye, they may interact. However, there is a risk that FRET will not be observed.
  • the evanescent field molecular imaging method is a method described in Funatsu, ⁇ ⁇ , et al., Nature, 374, 555-559 (1995), etc., in which molecules immobilized on a transparent body such as glass are used as a solution. This method involves contacting a second molecule, irradiating it with a light source such as a laser beam at an angle at which an evanescent field is generated, and measuring or analyzing the generated evanescent light with a detector. These operations can be performed using an evanescent field fluorescence microscope known per se.
  • either the C-terminal modified protein or the target molecule must be immobilized by the method described above.
  • the target molecule does not need to be modified when immobilized, but when used without being immobilized, it must be modified with the above-mentioned modifying substance.
  • a substrate for immobilizing the C-terminal modified protein or target molecule As a substrate for immobilizing the C-terminal modified protein or target molecule, A substrate made of a material such as a lath is used, and quartz glass is preferably used. Further, it is preferable that the surface is subjected to ultrasonic cleaning in order to prevent scattering of laser light.
  • the method of contacting the C-terminal modified protein or the modified target molecule, which is not immobilized, with the immobilized molecule in this method is not particularly limited as long as the two molecules are in contact with each other to an extent sufficient for interaction.
  • a solution in which the unmodified C-terminal modified protein or modified target molecule is dissolved at an appropriate concentration in a buffer commonly used in biochemistry is prepared, and this is immobilized on the surface of the solid phase.
  • the dropping method is preferred.
  • 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 proteins or modified target molecules and immobilizing the same on the substrate is used.
  • the fluorescence polarization method (Perran, J., et al., J. Phys. Rad., 1, 390-401 (1926)) states that a fluorescent molecule excited by fluorescence polarization changes its steady state during the excited state. If it is kept, it emits fluorescence in the same plane of polarization, but if the excited molecule performs rotational Brownian motion etc. during the excited state, the emitted fluorescence will be on a different plane from the excitation light It is a way to use that.
  • the motion of the molecule is affected by its size, and when the fluorescent molecule is a macromolecule, there is little motion of the molecule during the excited state, and the emitted light remains polarized.
  • the emitted light is depolarized because of the high speed of movement. 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, and the motility of this molecule and the state of its existence are determined from the ratio of the fluorescence intensity on both planes. Information can be obtained. According to this method, the behavior of the target molecule that interacts with the fluorescently-modified molecule can be tracked without being affected by contaminants. This is because the change in polarization is measured only when the target molecule interacts with the fluorescently modified molecule.
  • BECON manufactured by Panyera
  • the present method can also be performed by using these devices.
  • any method can be used to bring the target molecule into contact with the C-terminal modified protein, as long as the two molecules come into contact with each other to an extent sufficient to interact with each other.
  • the interaction between the C-terminally modified protein and the target molecule measured in this method may not necessarily be as specific as the antigen-antibody method, so to detect the optimal combination, It is effective to quantify the degree of action.
  • an index indicating the degree of interaction for example, a value of a minimum target substance concentration that gives a maximum fluorescence polarization degree for a certain concentration of a C-terminal modified protein can be used.
  • a large number of analyzes can be performed at the same time, for example, by introducing a plurality of different c-terminal modified proteins to each measurement well of the fluorescence polarization elimination measurement apparatus, and adding a specific target molecule solution Alternatively, a specific C-terminal modified protein is added, and a plurality of different target molecule solutions are added to each well.
  • Surface plasmon resonance is a method in which surface plasmons are excited by molecules interacting at the metal / 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 When measuring or analyzing the interaction between protein and target molecule using this method, the C-terminal modified protein must be immobilized by the method described above, but the target molecule must be modified. Absent.
  • a substrate for immobilizing the C-terminal modified protein a substrate in which a metal thin film of gold, silver, platinum or the like is formed on a transparent substrate of glass or the like is used.
  • the transparent substrate may be any substrate as long as it is generally used for a surface plasmon resonance device, and is generally made of glass or the like as being made of a material transparent to laser light.
  • the thickness is about 0.1 to 5 mm.
  • the thickness of the metal thin film is suitably about 100 to 200 OA.
  • a commercially available solid substrate for such a surface plasmon resonance device can also be used.
  • the C-terminal modified protein can be immobilized on the substrate by the method described above.
  • the method of bringing the target molecule into contact with the C-terminal modified protein in this method may be any method as long as the two molecules come into contact with each other to an extent sufficient to interact with each other.
  • a method may be used in which the solid-phased C-terminal protein is brought into contact with a solution dissolved at an appropriate concentration in a commonly used buffer solution.
  • a commercially available surface plasmon resonance device for example, BIAcore2000 (Pharmacia Biosensor). After bringing both molecules into contact with each other, the change in relative intensity of the reflected light with time is measured using a surface plasmon resonance device known per se, and the immobilized C-terminal modified protein and target are measured. Analyze molecular interactions.
  • a large number of analyzes can be performed simultaneously, for example, by immobilizing and immobilizing a plurality of C-terminal modified proteins on a substrate used in the surface plasmon resonance apparatus, or by using one type of solid phase.
  • a method of contacting a plurality of types of target molecules with the modified C-terminal modified protein is used.
  • Enzyme Linked Immunosorbent Assay Crowther, JR, Methods in Molecular Biology, 42 (1995)
  • a solution containing an antibody is contacted with an antigen immobilized on a solid phase.
  • the fluorescence emitted from the modified molecule such as IgG
  • the modified molecule that specifically binds the antibody remaining on the immobilized antigen to the antigen
  • ELISA reader a commercially available detector
  • the C-terminal modified protein serving as the antigen be immobilized by the above-described method. Further, the target molecule to be an antibody needs to be modified with the above-mentioned modifying substance.
  • a plastic microplate usually used for ELISA can also be used as a substrate for immobilizing a C-terminal modified protein serving as an antigen.
  • the method for bringing the modified target molecule, which is to be an antibody, into contact with the solid phase molecule in this method may be any method as long as the two molecules come into contact with each other to an extent sufficient for interaction.
  • a preferred method is to prepare a solution in which the molecule is dissolved at an appropriate concentration in a buffer commonly used in biochemistry and inject the solution 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, and the fluorescence emitted from the modified molecules remaining on the solid phase is measured.
  • a molecule that interacts with the immobilized antigen molecule can be identified by measurement or analysis using a commercially available ELISA reader or the like.
  • a target molecule that has been measured by each of the above methods and that has recognized an interaction with a C-terminal modified protein is known per se if the primary structure of the molecule is unknown.
  • the primary structure can be analyzed by an appropriate method. Specifically, when the target molecule for which the interaction is recognized is a protein, the primary structure can be specified 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 automatic DNA sequencer or the like.
  • a known suitable device for immobilizing a C-terminal modified protein on a solid phase via a modified portion described above is used.
  • An apparatus can be constructed by combining various means. Each means in the present apparatus is known, and the operations such as holding the substrate, adding the C-terminal modified protein solution, and washing in these means may be performed by methods 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 protein-target molecule interaction measurement described above.
  • Each means in the present apparatus is itself known, and operations such as substrate holding, addition of target 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 measuring protein-target molecule interaction can be constructed.
  • Example 1 Preparation of Translation Template and Translation (C-Terminal Labeling)
  • the coding molecule was C-jun or C-fos derived from mouse (Gentz R, Rauscher FJ 3d, Abate C, Curran T (1989) Science 243: 1695).
  • MA templates were transcribed (37 ° C, 2h) using RiboMAX TM Large Scale RNA Production Systems (Promega), the synthesized mHNA was purified using RNeasy Mini Kits (QIAGEN), and the mMA template (coding molecule) was purified. Obtained.
  • FIG. 6 shows that the amount of translation increases when the protein has the SNNS sequence (X sequence) rather than the poly A sequence (A sequence) and further has the SNNS-poly A sequence (XA sequence). From this, it can be said that a translation template having an XA sequence is preferable for general translation and C-terminal labeling. Also, from the translation result when the X sequence is changed, the X sequence must be composed of at least 4 bases in order to exhibit the effect in combination with the A sequence, and the first and fourth bases are C Or G, indicating that an SNNS (S is C or G) configuration is required.
  • a translation template was prepared by ligating one molecule of PEG spacer (see Production Examples 1 to 4 below) to the 3 ′ side of the mRNA template.
  • the amount of translation can be increased by ligating a part of the PEG linker to basically any sequence of the coding section (A in FIG. 7).
  • the translation template (XA) which has a coding part with an XA sequence, has about 3 to 4 times more translation than the code molecule (None), which has neither the XA sequence nor any PEG spacer. was shown.
  • the PEG4000Puro-Boc configuration maximized the translation amount in the code portion having the XA sequence (B in FIG. 7).
  • a translation template having an XA sequence in which a part of the PEG spacer is ligated is suitable for general translation and C-terminal labeling. From Fig. 8, the translation amount of the coding molecule having the XA sequence ( ⁇ ; XA) and the coding molecule having no XA sequence (mouth; None) reached saturation in 3 hours, but the translation template ( ⁇ ; PEG4000Puro-Boc) XA + PEG400Pm? O-Boc) showed an increase in translation even at 6 hours (A in FIG. 8).
  • the translation template ( ⁇ ) having PEG4000Puro-Boc is about twice as large as the code molecule having the XA sequence (A; XA) and the coding molecule having no XA sequence. (Mouth; None) (Fig. 8A).
  • a coding molecule and a translation template with a PEG spacer In comparison, the stability of the coding molecule (K; XA) was reduced by 50% in its mRNA in 1 hour, whereas that of the translation template with a PEG spacer part ( ⁇ ) in 13 hours. The rapid decrease of 50% indicates that the stability of the code molecule ( ⁇ ) having a part of the PEG spacer is very good (B in Fig. 8). From the above, it is considered that the increase in the amount of translation of the translation template ( ⁇ ) is due to the improved stability of the ligated PEG4000Puro-Bo.
  • a PEG spacer molecule (see Production Examples 1 to 4) ligated to a coding molecule (mRNA template) was used as a translation template.
  • mRNA template Jun-XA, Jun-A
  • PEG 2000 Puro a part of PEG spacer
  • the ligated mA template was translated in the same manner as in Example 1 using Wheat Germ Extract (Promega) as a cell-free translation system for wheat germ, and the corresponding molecule was converted to 8 M urea 10% SDS-PAGE. And detected by fluorescence (fluorescein) using a multi-image analyzer, Molecular Imager FX (Bio-Rad).
  • the amount of free protein was determined by labeling the protein using Fluor-dCpPuro as a modifier at the same time as translation (Miyamoto-Sato, R, Nemoto, ⁇ , Kobayashi, ⁇ , and Yanagawa, H. 2000). Nucleic Acids Res. 28: 1176-1182; Nemoto, N., Miyamoto-Sato, E. and Yanagawa, H. (1999) FEBS Lett., 462: 43-46) and 8 M urea 10% SDS- Electrophoresis by PAGE and 15% SDS-PAGE, and the fluorescence (fluorescein) of the band was measured with a multi-image analyzer, Molecular Imager FX (Bio-Rad). In addition, Western plot using T7-tag antibody The total protein was determined with. Fig. 9 shows a graph summarizing the results.
  • PEG spacer molecule (U) was synthesized by the method shown in FIG. 11 using the reagent shown in FIG.
  • the amidite reagents (1-5) in Figure 10 were purchased from Glen Research (America, Virginia). PEG having an average molecular weight of 1,000, 2000, and 3000 was purchased from NOF Corporation (Shibuya-ku, Tokyo). PEG having an average molecular weight of 4000 was purchased from Hurikisha (Switzerland).
  • the amidite reagent (6) was synthesized from these starting materials using the method reported by Jaschke et al. (Jaschke, A. et al. (1993) Tetrahedron Lett. 34: 301-304). 10 in FIG.
  • DMTr in FIG. 10 represents a 4,4, -dimethoxytrityl group
  • Fmoc in FIG. 11 represents a 9-fluorenemethoxycarbonyl group.
  • the PEG spacer single molecule (14) was synthesized by the method shown in FIGS. 11 and 12 using the reagent shown in FIG.
  • rhodamine green (RhodG) active ester (7) was obtained from Morexura Probes (Oregon, USA), and Cy5 active ester (8) and Cy3 active ester (9) were obtained from Amersham Pharmacia Biotech ( Igi (Squirrel, Buckinghamshire).
  • FIG. 12 was synthesized by applying the method reported by Ikeda et al. (Ikeda, S. et al. (1998) Tetrahedron Lett. 39: 5975-5978).
  • Boc represents a tert-butoxycarbonyl group.
  • Boc-protected PEG spacer part (15) was converted from 12 (400 ig, containing puromycin lO ⁇ mol) using the same method as PEG spacer part (11). Synthesis did. The structure and yield of the obtained Boc-protected PEG spacer single molecule (15) are shown below. p (dCp) 2 T (Fl) pPEG (4000) p (dCp) 2 Puro (Boc) Yield 41% Production Example 4 Synthesis of puromycin-free PEG spacer single molecule (16)
  • Modifiers 1 to 7 were synthesized using the reagents shown in FIGS. 15 and 16 and the method outlined in FIGS. 17 and 18.
  • amidite reagents (15-19) were purchased from Glen Research (American, Virginia), and succinimide reagents (20-22) were purchased from Pierce (Illinois, USA).
  • Modifier 1 yield 44%, UV (50% MeOH -H 2 0) e max 558 nm; MS m / z 2037 (M - H) one modifying agent 2: Yield 32%, UV (H 2 0 ) Belly 558 nm; MS m / z 2035 (MH)
  • Modifier 3 Yield 83 ⁇ 4 ⁇ UV (H 2 0) Aniax 506 nm; MS m / z 2093 (M-H) —
  • the stability of the translation template in the translation system is improved, and the absolute amount of the translation is increased.
  • the ratio of the target assigning molecule to the total protein produced by translation is improved, and the assigning molecule is efficiently obtained. be able to.

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Abstract

L'invention concerne des matrices de traduction qui permettent de synthétiser efficacement des protéines et qui peuvent constituer un outil utile pour les études post-génomiques à haut rendement pour analyser des structures et des fonctions. Une matrice de translation est composée d'une partie codante et éventuellement d'une partie PEG. Si elle comprend une région receveuse à l'extrémité 3' de la partie codante et une séquence d'amélioration de la traduction contenant la séquence SNNS dans la partie amont 5' de la région receveuse, cette partie PEG, qui ne peut pas être liée à une protéine au moyen d'une réaction de transpeptidation, peut être avantageusement utilisée dans la synthèse des protéines. Si elle comprend une région receveuse à l'extrémité 3' de la partie codante, cette partie PEG, qui ne peut pas être liée à une protéine au moyen d'une réaction de transpeptidation, peut être avantageusement utilisée dans la localisation. L'invention concerne également des protéines synthétisées au moyen de ces matrices de traduction, de protéines modifiées à extrémité C et de molécules localisées. Il est également possible de conférer une propriété de modification fonctionnelle à des modificateurs de ces protéines modifiées à extrémité C et aux parties PEG des molécules localisées.
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WO2016143799A1 (fr) * 2015-03-09 2016-09-15 国立大学法人名古屋大学 Activateur de traduction pour l'utilisation dans un système de synthèse de protéine sans cellule et son utilisation
WO2023008337A1 (fr) 2021-07-26 2023-02-02 ピューロテックバイオ株式会社 Agent anti-virus de l'hépatite b ciblant le facteur hôte lipg

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JP4723713B2 (ja) * 2000-09-25 2011-07-13 トヨタ自動車株式会社 ｍＲＮＡの潜在的翻訳制御因子のスクリーニング方法
JP4030019B2 (ja) * 2001-12-07 2008-01-09 学校法人慶應義塾 対応付け分子とｃ末端ラベル化蛋白質の複合体および対応付け分子の複合体、ならびにそれらの複合体を利用した蛋白質間相互作用解析方法

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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
WO2002048347A1 (fr) * 2000-12-14 2002-06-20 Keio University Molecule correspondant a un genotype et a un phenotype, elements en etant faits et ses procedes d'obtention et d'utilisation

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WO2016143799A1 (fr) * 2015-03-09 2016-09-15 国立大学法人名古屋大学 Activateur de traduction pour l'utilisation dans un système de synthèse de protéine sans cellule et son utilisation
JPWO2016143799A1 (ja) * 2015-03-09 2017-11-16 国立大学法人名古屋大学 無細胞タンパク質合成系に用いるための翻訳促進剤及びその利用
WO2023008337A1 (fr) 2021-07-26 2023-02-02 ピューロテックバイオ株式会社 Agent anti-virus de l'hépatite b ciblant le facteur hôte lipg

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