WO2017209280A1 - Nouveau procédé d'identification de protéine au moyen de levure et banque d'adnc fragmentés - Google Patents

Nouveau procédé d'identification de protéine au moyen de levure et banque d'adnc fragmentés Download PDF

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Publication number
WO2017209280A1
WO2017209280A1 PCT/JP2017/020614 JP2017020614W WO2017209280A1 WO 2017209280 A1 WO2017209280 A1 WO 2017209280A1 JP 2017020614 W JP2017020614 W JP 2017020614W WO 2017209280 A1 WO2017209280 A1 WO 2017209280A1
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Prior art keywords
protein
cdna
yeast
gene encoding
vector
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PCT/JP2017/020614
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English (en)
Japanese (ja)
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昭世 岸田
小山　浩史
想子 岸田
幹雄 飯島
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国立大学法人鹿児島大学
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Priority to JP2018521014A priority Critical patent/JP6998056B2/ja
Publication of WO2017209280A1 publication Critical patent/WO2017209280A1/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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

Definitions

  • the present invention relates to a protein identification method using, for example, yeast and a fragmented cDNA library.
  • the yeast two-hybrid method is known as a method for identifying binding of intracellular (cytoplasmic) molecules (Non-Patent Documents 1 to 3).
  • the yeast two-hybrid method uses a gene vector to add a known gene to which a nuclear translocation signal has been added and a full-length cDNA chimeric gene to which the nuclear translocation signal has been added (usually reverse transcribed with an oligo dT primer in a commercial product)
  • the types are expressed and a bond between molecules is formed in the nucleus, the bond between the molecules is detected by the activity of the reporter gene whose expression is induced, light emission, or the like.
  • yeast two-hybrid method when a gene encoding a membrane protein such as a ligand molecule or a receptor is inserted into a vector, the chimeric gene is nucleated by expression of the signal peptide at the 5 'end or the transmembrane region near the 3' end.
  • the yeast two-hybrid method is not suitable for analysis of ligand-receptor binding.
  • yeast two-hybrid method many modifications have been reported.
  • the cells to be used can be changed to organisms other than yeast, or can be improved by developing a reporter gene portion.
  • a modification of the yeast two-hybrid method which is intended to be detected in the nucleus as a partial region library of (membrane) protein and to detect the interaction that originally occurs outside the nucleus, is known. There wasn't.
  • the conventional yeast two-hybrid method is not suitable for analysis of ligand-receptor binding due to the expression of the signal peptide at the 5 ′ end and the transmembrane region near the 3 ′ end. Therefore, the present invention aims to provide a method suitable for analyzing the binding between proteins containing a ligand-receptor by modifying the yeast two-hybrid method.
  • the present invention includes the following.
  • a protein identification method comprising a step of culturing a yeast transformant into which the first and second vectors have been introduced, wherein the first vector comprises a gene encoding a nuclear translocation signal and a DNA binding protein
  • a gene encoding a decoy protein and a second vector comprising a gene encoding a nuclear translocation signal, a gene encoding a transcriptional activation protein, and a fragmented partial cDNA
  • the fragmented partial cDNA encodes a prey protein lacking a signal peptide, a cell membrane or organelle localization sequence or a transmembrane region, and in the nucleus of the yeast transformant,
  • a fusion protein comprising a translocation signal, a DNA binding protein and a decoy protein, and said translocation signal and transcription-activating protein Fusion protein was expressed containing the prey proteins encoded by fragmented partial cDNA, the binding of the bait protein and ⁇ product protein, detecting the activity of
  • a vector comprising a gene encoding a nuclear translocation signal, a gene encoding a transcriptional activation protein, and a fragmented partial cDNA, wherein the fragmented partial cDNA is described in (4) or (5) The vector, which is cDNA.
  • the present invention it is possible to contribute to the elucidation of signal molecules involved in various diseases by elucidating the receptors for orphan ligands and searching for and identifying molecules to which membrane proteins bind.
  • the protein identification method according to the present invention includes a step of culturing a yeast transformant into which the first and second vectors have been introduced, wherein
  • the first vector includes a gene encoding a nuclear translocation signal, a gene encoding a DNA binding protein, and a gene encoding a bait protein
  • the second vector includes a gene encoding a nuclear translocation signal, a gene encoding a transcriptional activation protein, and a fragmented partial cDNA.
  • the fragmented partial cDNA is a signal peptide, a cell membrane, or an organelle.
  • a fusion protein comprising the nuclear translocation signal, a DNA binding protein, and a decoy protein, and a prey encoded by the partial cDNA fragmented with the nuclear translocation signal, the transcriptional activation protein A fusion protein containing a protein is expressed, and the binding between the decoy protein and the prey protein is detected using the activity of a reporter gene as an indicator, Is the method.
  • a fusion protein of a DNA-binding protein and a molecule of interest (bait protein) and a cDNA library (derived from) a transcription library and a full-length cDNA (prey protein)
  • a cDNA library derived from) a transcription library and a full-length cDNA (prey protein)
  • the activity of the reporter gene that induces expression is used as an index to bind the decoy protein to the library-derived molecule. Is detected.
  • the yeast two-hybrid method has become widespread as an inexpensive large-scale screening technique using the binding of two molecules as an index.
  • a protein derived from a cDNA library contains a signal peptide, a sequence that localizes to cell membranes or various organelles, the molecule cannot be expressed in the nucleus, and a bond with a decoy protein is formed in the nucleus. And reporter gene expression cannot be detected.
  • the yeast two-hybrid method is generally used as a technique for detecting the binding between intracellular proteins.
  • a library commercially available in the prior art is one in which a cDNA that has been reverse-transcribed and synthesized from the 3 ′ end of mRNA using an oligoolidT primer (substantially full length) is inserted.
  • this method uses a cDNA library with intentionally fragmented cDNA inserted, partial regions of molecules including membrane proteins and various localized sequences that were missed in the conventional method are expressed in the yeast nucleus. By doing so, binding to a decoy protein can be detected, and ligand-receptor binding that usually occurs outside the cell and association of membrane proteins can be detected inside the yeast nucleus, both inside and outside the cell.
  • first, first and second vectors are prepared.
  • the first vector includes a fusion gene in which a gene encoding a nuclear translocation signal, a gene encoding a DNA binding protein, and a gene encoding a decoy protein are functionally linked.
  • the first vector may be any vector that functions in yeast, and examples thereof include pBTM116, pGBKT7, and pGBT9.
  • a nuclear transfer signal is sent.
  • Encoding gene is included in the fusion gene or added in advance (eg, Large T antigen residues 47 to 54 (PKKKRKVE: SEQ ID NO: 1), LexA protein endogenous nuclear localization signal sequence (KRLKK: SEQ ID NO: 2), etc.) Must have been.
  • DNA binding protein examples include a DNA binding domain (DBD) of Gal4 protein, a DBD of LexA protein, and the like.
  • DBD DNA binding domain
  • decoy proteins include orphan ligands whose receptors are unknown, proteins encoded by partial cDNAs excluding various localization signals (such as transmembrane domains and signal sequences localized to organelles) Is mentioned.
  • each gene is included in a functionally linked form in order to express a fusion protein including a nuclear translocation signal-DBD-bait protein.
  • the second vector includes a fusion gene in which a gene encoding a nuclear translocation signal, a gene encoding a transcription activation protein, and a fragmented partial cDNA are functionally linked.
  • the second vector may be any vector that functions in yeast, and examples thereof include pACT2, pGADT7, and pGAD10.
  • the transfer signal in order to transfer the fusion protein encoded by the fusion gene comprising the gene encoding the transcriptional activation protein and the fragmented partial cDNA into the yeast nucleus,
  • the transfer signal must be endogenous or previously added (for example, Large T antigen residuesres47 to 54 (PKKKRKVE: SEQ ID NO: 1), LexA protein endogenous nuclear localization signal sequence (KRLKK: SEQ ID NO: 2), etc.) Don't be.
  • the transcription activation protein includes a DNA binding protein encoded by the fusion gene contained in the first vector and transcription activity when located close to a reporter gene control sequence (promoter etc.) in yeast. And activates transcription of a reporter gene, such as an activator domain (AD) of Gal4 protein corresponding to DBD of Gal4 protein.
  • a reporter gene control sequence promoter etc.
  • AD activator domain
  • the fragmented partial cDNA is different from a full-length gene cDNA library prepared using an oligo dT primer, for example, a known cDNA library preparation (e.g., cDNA Library Construction Kit (Takara Bio Inc.)).
  • a known cDNA library preparation e.g., cDNA Library Construction Kit (Takara Bio Inc.)
  • a random primer for example, a random primer comprising the base sequence described in SEQ ID NO: 3 used in the following Examples
  • the cDNA obtained by random prime reverse transcription has, for example, a length of 100 to 1800 bp (preferably 100 to 1000 bp).
  • a cDNA library fragmented by cleavage using sonication or the like is prepared, for example, by adding an EcoRI adapter to the end and binding it to a large excess of vector having an EcoRI stump.
  • Such partial cDNAs include those encoding a prey protein that binds to a ligand or membrane protein, lacking a signal peptide, a cell membrane or organelle localization sequence or a transmembrane region. Examples of the prey protein include a receptor protein.
  • a fusion protein containing a nuclear protein a prey protein encoded by a Gal4 activator domain (AD) -fragmented cDNA is sequentially expressed.
  • AD Gal4 activator domain
  • the first and second vectors are then transformed into yeast to produce yeast transformants that express the first and second vectors.
  • yeast include budding yeast (Saccharomyces cerevisiae).
  • transcription is activated by the DNA binding protein encoded by the fusion gene contained in the first vector and the transcription activation protein encoded by the fusion gene contained in the second vector.
  • control sequences to be converted and a reporter gene located downstream thereof include a base sequence called UASG (UpstreamUpActivation Sequences for galactose) for the Gal4 protein.
  • the reporter gene include a gene encoding ⁇ -galactosidase and HIS3. The control sequence and the reporter gene located downstream thereof are present in the yeast nucleus because they are integrated into the genomic DNA.
  • the method for introducing the first and second vectors into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.
  • the obtained yeast transformant is cultured under conditions that allow it to grow.
  • a fusion protein comprising a nuclear translocation signal, a DNA-binding protein and a decoy protein, and a prey protein encoded by the nuclear translocation signal, a transcriptional activation protein and a fragmented partial cDNA
  • the decoy protein and the prey protein interact with each other so that the DNA-binding protein and the transcriptional activation protein are close to each other and bind to a control sequence located upstream of the reporter gene. , Transcription of the reporter gene is promoted. Therefore, the binding of the decoy protein and the prey protein can be confirmed using the activity of the reporter gene as an index, and the prey protein corresponding to the decoy protein can be identified.
  • the temperature is set to, for example, 25 to 30 ° C.
  • the pH of the medium is, for example, pH 5 so that the transformants grow and the reporter protein encoded by the reporter gene is not inactivated. Set at around .8 and continue culturing until the required amount of cells for each assay is obtained.
  • the gene fragment encoding the partial prey protein can be identified by determining the sequence of the fragmented partial cDNA contained in the second vector. . Furthermore, by comparing the gene fragment encoding the identified partial prey protein with, for example, a gene sequence registered in a known database, the gene encoding the prey protein (if registered) or the gene concerned The full-length prey protein encoded by can also be identified.
  • the detection sensitivity of binding up to about 1 ⁇ M Kd binding is expected to be detected by using the yeast reporter gene system, compared to the case of panning in cultured cells.
  • Improved S / N ratio, faster screening (normally about 14 days are required to determine the binding of about 1 million clones), stability of recovered genes, faster cloning, economy, etc. Be expected.
  • receptor identification of orphan ligands may be directly linked to drug discovery, and this method can be applied to receptor identification of orphan ligands.
  • the present method can be widely applied to the binding screening between membrane proteins.
  • the present invention also relates to a reagent kit for the present method comprising the cDNA itself obtained by the above-mentioned random prime reverse transcription, the second vector itself, the cDNA obtained by the random prime reverse transcription or the second vector.
  • the reagent kit can further include, for example, a buffer and a container used in the present method, instructions for use, and the like.
  • reverse transcriptase (PrimeScript RTase Takara Bio Inc.) (200 U / ⁇ l) 1 ⁇ l, RNase inhibitor (40 U / ⁇ l) 1 ⁇ l, reaction buffer (250 mM Tris-HCl (pH 8.3 ), 375 mM KCl, 15 mM MgCl 2 ) 4 ⁇ l and RNase-free water 4 ⁇ l were added to make 20 ⁇ l, and a reverse transcription reaction was performed at 42 ° C. for 1 hour. After 1 hour, it was cooled on ice for 2 minutes. The cDNA synthesized by this reaction was designated as 1st strand cDNA.
  • 2nd strand cDNA synthesis 1st strand cDNA is mixed with dNTP mixture (dATP, dCTP, dGTP, dTTP) 4.5 ⁇ l, RNaseH and DNA ligase mixture 2 ⁇ l, DNA polymerase I (20 U / ⁇ l) 2 ⁇ l, 30 ⁇ l of reaction buffer and 87.5 ⁇ l of RNase-free water were added to make 146 ⁇ l, followed by reaction at 16 ° C. for 2 hours. After 2 hours, the mixture was allowed to stand at 70 ° C. for 10 minutes and then allowed to stand at room temperature for 5 minutes or more. The double-stranded cDNA synthesized by this reaction was designated as 2nd strand cDNA.
  • dNTP mixture dATP, dCTP, dGTP, dTTP
  • RNaseH and DNA ligase mixture 2 ⁇ l
  • DNA polymerase I 20 U / ⁇ l
  • reaction buffer 30 ⁇ l
  • the XhoI recognition sequence in the random primer is cleaved, but the XhoI recognition sequence in the cDNA is not cleaved by XhoI because 5-methyl dCTP is used during the synthesis of the first strand cDNA.
  • the vector and EcoRI-XhoI-cleaved cDNA combined were introduced into E. coli by electroporation, and any 10 clones were selected and analyzed for the size of the fragmented cDNA inserted by PCR. After confirming that it was 100 to 1000 bases, a cDNA library was recovered from E. coli.
  • the vector pBTM116-HA-KM-hENHO contains an endogenous nuclear localization signal sequence (KRLKK: SEQ ID NO: 2) and DNA binding domain (DBD) of the LexA protein in order from the 5 ′ end to the 3 ′ end. It has a gene that encodes it and a gene that encodes human adropin (hENHO) as a gene that encodes a decoy protein.
  • Saccharomyces cerevisiae L40 has a gene (reporter gene) encoding ⁇ -galactosidase and an HIS3 gene under the control of UASG downstream. When molecules expressed from the two plasmids are combined in the cell, yeast cells can grow even in a His-deficient medium due to the expression of the HIS3 gene.
  • the seeded medium was transferred to a 30 ° C. incubator and cultured for several days until colonies were visible. Colonies were transferred to fresh SD-Leu-Trp medium and cultured in a 30 ° C. incubator. Using a part of the grown yeast, ⁇ -galactosidase assay was performed, and a group of clones positive for ⁇ -galactosidase activity was analyzed. As a result, those containing partial cDNA of membrane protein were included.

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Abstract

La présente invention vise à fournir un nouveau procédé d'identification d'une protéine dans un noyau de levure. Plus précisément, la présente invention concerne un procédé d'identification de protéine qui consiste : dans un noyau de levure, à exprimer une protéine de fusion comprenant un signal de localisation nucléaire, une protéine de liaison à l'ADN et une protéine appât, et à exprimer une autre protéine de fusion comprenant un signal de localisation nucléaire, une protéine d'activation transcriptionnelle et une protéine proie codée par un ADNc partiel fragmenté, ladite protéine proie étant dépourvue de peptide signal, une séquence de localisation d'organite intracellulaire ou de membrane cellulaire ou un domaine transmembranaire ; puis à détecter la liaison de la protéine appât à la protéine proie au moyen de l'activité, de la luminescence, etc. d'un gène rapporteur en tant qu'indication.
PCT/JP2017/020614 2016-06-03 2017-06-02 Nouveau procédé d'identification de protéine au moyen de levure et banque d'adnc fragmentés WO2017209280A1 (fr)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2002079493A2 (fr) * 2001-03-29 2002-10-10 Hybrigen, Inc. Librairies de genes hybrides ameliorees et utilisations correspondantes
JP2012523836A (ja) * 2009-04-17 2012-10-11 ニューヨーク ユニバーシティ Ｔｎｆファミリー受容体を標的とし、ｔｎｆ作用を拮抗するペプチド、その組成物、方法および使用

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JPWO2005050518A1 (ja) 2003-11-20 2007-12-06 学校法人慶應義塾 遺伝子および／又は蛋白質のデータベースを用いた相互作用マップの作成方法、ならびに、それを実現するためのソフトウエアおよび装置

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WO2002079493A2 (fr) * 2001-03-29 2002-10-10 Hybrigen, Inc. Librairies de genes hybrides ameliorees et utilisations correspondantes
JP2012523836A (ja) * 2009-04-17 2012-10-11 ニューヨーク ユニバーシティ Ｔｎｆファミリー受容体を標的とし、ｔｎｆ作用を拮抗するペプチド、その組成物、方法および使用

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