WO2011162355A1 - 蛋白質ライブラリ調製を目的としたオリゴヌクレオチドライブラリの高速成熟化法 - Google Patents
蛋白質ライブラリ調製を目的としたオリゴヌクレオチドライブラリの高速成熟化法 Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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Definitions
- the present invention relates to a method for rapid maturation of an oligonucleotide library for the purpose of preparing a protein library.
- oligonucleotides When introducing a plurality of random amino acid sequences at a certain site of a protein, randomly synthesized oligonucleotides are introduced into the protein gene. However, when the introduced sequence is synthesized completely at random (NNN sequence), stop codons appear at a rate of about 1/21. Moreover, when the synthetic oligonucleotide to be introduced is a long chain, oligonucleotides with bases deleted or inserted are mixed at a rate of several percent. All of these cause a significant reduction in the diversity of the protein library that is obtained, so it is necessary to remove these oligonucleotides when preparing the oligonucleotide library.
- a method of removing these unnecessary oligonucleotides from the synthetic oligonucleotide library there is a method using drug selection of antibiotics.
- a random oligonucleotide library to be matured is linked to the 5 'end of a drug resistance gene such as ⁇ -lactamase and introduced into a plasmid.
- a drug resistance gene such as ⁇ -lactamase
- this plasmid is transformed into E. coli and then applied to an agar plate containing the corresponding antibiotic, only E. coli with an in-frame gene that does not contain a frame shift due to deletion or insertion of a stop codon, base, or Since it can be expressed, it can grow as a colony on a plate. Therefore, by recovering genes from these colonies, a matured oligonucleotide library free from unnecessary oligonucleotides can be obtained.
- the time required for the inventors to mature an oligonucleotide library with 10 8 diversity by this method was about 1 week. If an oligonucleotide library that encodes an antibody gene library in which six CDRs (complementarity determining regions) of an antibody are randomized is created, maturation is sequentially performed on the oligonucleotide library that encodes each of these six CDRs. Therefore, it takes about 6 weeks to mature the entire oligonucleotide library encoding the antibody gene library.
- Patent Document 1 discloses ribosome display.
- Patent Document 1 paragraph [0051] discloses an mRNA in which a sequence encoding a translation reaction elongation stop sequence of Escherichia coli SecM is arranged downstream of a spacer sequence.
- An object of the present invention is to provide a method for producing a matured oligonucleotide library capable of producing many types of matured oligonucleotide libraries at once in a short time with ease and at low cost.
- Another object of the present invention is to provide an efficient maturation method for a random oligonucleotide library using ribosome display of an in vitro selection system (Japanese Patent Laid-Open No. 2008-271903).
- the in-frame rate is improved by adding an arrest sequence (for example, a SecM sequence) to the 3 ′ end of the oligonucleotide, so that maturation can be achieved very efficiently. It is based on the knowledge that.
- an arrest sequence for example, a SecM sequence
- the first aspect of the present invention relates to a method for producing a matured oligonucleotide library.
- a tag sequence is added to the 5 ′ end of the oligonucleotide library to be matured, and an arrest sequence (for example, a SecM sequence) is added to the 3 ′ end. (Terminal modified sequence) is obtained.
- the oligonucleotide library to be matured is a random oligonucleotide library.
- An example of a random oligonucleotide library is an oligonucleotide library containing NNS sequences.
- An example of an NNS sequence is an NNS sequence that includes a codon corresponding to 31 amino acids.
- the random oligonucleotide library to be matured is a complete random oligonucleotide library, stop codons appear at a rate of about 1/21. For this reason, the random oligonucleotide library preferably does not contain a complete random sequence (NNN).
- the end-modified sequence is transcribed to obtain a transcript.
- the transcript is then translated in vitro.
- an example of a tag sequence is a FLAG sequence.
- the product can be easily recovered by using beads on which an anti-FLAG antibody is immobilized.
- a cell-free translation system it is preferable to translate the transcript in vitro using a cell-free translation system. Since the treatment is performed completely in vitro, the maturation efficiency of the oligonucleotide library can be improved.
- An example of a cell-free translation system is the E. coli cell-free translation system.
- a preferable cell-free translation system is a so-called PURE system which is a reconfigurable cell-free translation system.
- the reconstituted cell-free protein synthesis system is a synthesis system consisting only of specified factors such as translation factors and ribosomes involved in protein synthesis developed by a group including the present inventors.
- Ribosome display is a method of selecting a protein encoding a polypeptide having a specific function by forming an mRNA-ribosome-oligopeptide ternary complex (ribosome display complex) in an in vitro translation system.
- the terminal modification product of the present invention has an arrest sequence at the 3 'end of the oligonucleotide library to be matured.
- the arrest sequence is not normally translated during translation.
- oligonucleotide libraries having great diversity for example, 10 11 to 10 12 types or more
- the method for producing a matured oligonucleotide library of the present invention is simple and efficient in operation, for example, 8 types of oligonucleotide libraries to be matured independently prepared can be matured simultaneously in one day. Can be This is a significant difference compared to the conventional method that required about one week for one type of maturation.
- the method for producing a matured oligonucleotide library of the present invention does not require a large amount of expensive enzymes, the cost can be reduced at the same time.
- FIG. 1 shows a random oligonucleotide library (NNS sequence) before and after maturation.
- the first aspect of the present invention relates to a method for producing a matured oligonucleotide library.
- a tag sequence is added to the 5 ′ end of the oligonucleotide library to be matured, and an arrest sequence (eg, SecM sequence) is added to the 3 ′ end.
- an arrest sequence eg, SecM sequence
- “maturation” refers to removal of an oligonucleotide including a frame codon caused by a stop codon or base deletion or insertion from an oligonucleotide library.
- the “maturation target oligonucleotide library” means a mixture of oligonucleotides that can be matured.
- Random oligonucleotide library means a mixture of oligonucleotides having various sequences, as defined below.
- “Maturated oligonucleotide library” means a mixture of matured oligonucleotides.
- An example of the oligonucleotide library to be matured is a random oligonucleotide library.
- An example of a random oligonucleotide library is an oligonucleotide library containing NNS sequences.
- An example of an NNS sequence is an NNS sequence containing 31 amino acid codons.
- stop codons appear at a rate of about 1/21.
- the random oligonucleotide library preferably does not contain a complete random sequence (NNN). Examples of such random sequences (incomplete random sequences) are NNK sequences, NNS sequences and NNY sequences.
- N means any one of adenine (A), guanine (G), cytosine (C), and thymine (T).
- K means either guanine (G) or thymine (T).
- S means either cytosine (C) or guanine (G).
- Y means either cytosine (C) or thymine (T).
- NK sequence refers to an oligonucleotide sequence containing a plurality of “NNK” in succession (that is, “NNK” ⁇ m (m is an integer of 2 or more)).
- NNS sequence refers to an oligonucleotide sequence containing a plurality of “NNS” in succession (that is, “NNS” ⁇ m (m is an integer of 2 or more)).
- NNY sequence refers to an oligonucleotide sequence containing a plurality of “NNY” in succession (ie, “NNY” ⁇ m (m is an integer of 2 or more)).
- the random oligonucleotide library is an oligonucleotide library that repeatedly contains NNK, NNS, or NNY sequences.
- oligonucleotide containing NNK sequence, NNS sequence or NNY sequence repeatedly means an oligonucleotide containing a plurality of random sequences selected from the group consisting of NNK sequence, NNS sequence and NNY sequence in one oligonucleotide.
- a plurality of NNK sequences, NNS sequences, or NNY sequences may be included, or a plurality of different random sequences may be included so as to include both NNK sequences and NNS sequences.
- Examples of the oligonucleotide constituting the random oligonucleotide library in the present specification include those having 3 ⁇ n bases.
- n is 5 or more and 20 or less, and may be 7 or more and 11 or less.
- a specific example of n is 9.
- an example of the tag sequence added to the 5 'end of the oligonucleotide library to be matured is a FLAG sequence.
- the tag sequence is not limited to the FLAG sequence.
- Another example of a tag array is a mick array.
- Antibodies that specifically bind to the FLAG tag and Mick tag are already on the market. For this reason, the protein which fuse
- the ribosome display complex containing the matured oligonucleotide library of the present invention has a FLAG tag, the ribosome display complex can be easily recovered and purified by using beads immobilized with an anti-FLAG antibody. .
- Arrest sequence is a sequence that stops translation of ribosome in the middle.
- Examples of arrest sequences derived from E. coli include SecM sequences (Nakatogawa and Ito (2002) Cell, vol. 108, p. 629-636), and TnaC sequences (Gong et al, (2002) c Science, vol. 297, p. 1864-1867).
- examples of arrest sequences for artificially synthesized Escherichia coli include the sequences reported by Turner et al. (Tanner et al., (2009) J. Biol. Chem, vol. 284, p. 34809-34818).
- an example of an arrest sequence derived from a eukaryote is a uORF sequence (Hood et al., (2009) Annu. Rev. Microbiol, vol. 63, p. 385-409).
- the end-modified sequence is transcribed to obtain a transcript.
- the transcription process is known in the field of biotechnology. Therefore, the transfer process can be performed based on a known method.
- the transcript is translated in vitro.
- An example of this translation step is to translate the transcript in vitro using a cell-free translation system.
- the maturation efficiency of the oligonucleotide can be improved by performing the treatment completely in vitro.
- an example of a preferred cell-free translation system is the E. coli cell-free translation system.
- a cell-free translation system is a so-called PURE system, which is a reconstituted cell-free translation system (for example, “Shimizu Y, Inoue A, Tomari Y, Suzuki T, k Yokogawa T, Nishikawa K, Ueda T, 2001) Cell-free transition reconstituted with purified components Nature Biotechnology 19, 751-755 "
- the PURE system is a cell-free translation system in which factors necessary for translation are individually prepared and reconfigured.
- the PURE system there is almost no contamination with nuclease or protease that causes a decrease in efficiency in performing ribosome display. For this reason, it has been reported that the use of the PURE system has a higher selection efficiency than the use of the cell-extraction-type cell-free translation system (Villemagne et al., 2006 (2006) J. Immunol. Methods, vol. 313, p. 140-148).
- many organisms are equipped with means to avoid the translational stop of ribosome that occurs during translation. For example, in the case of E.
- the PURE system that does not include the avoidance component described above is an optimal cell-free translation system.
- the cell-free translation system has an energy regeneration system and at least one amino acid.
- the energy regeneration system means an element related to regeneration of energy sources such as ATP and GTP necessary for protein synthesis. Examples of energy regeneration substances are enzymes involved in ATP regeneration (creatine kinase, pyruvate kinase, etc.) and their substrates (creatine phosphate, phosphoenolpyruvate, etc.).
- the cell-free translation system comprises at least one amino acid, preferably 20 naturally occurring amino acids.
- the cell-free translation system may further contain an unnatural amino acid.
- Cell-free translation systems include, for example, buffers (for example, HEPES potassium and trisacetic acid), various salts, surfactants, RNA polymerases (T7, T3, and SP6 RNA polymerases), chaperone proteins (DnaJ, DnaK, GroE). , GroEL, GroES, and HSP70), RNA (mRNA, tRNA, etc.), protease inhibitor, or (ribo) nuclease inhibitor.
- buffers for example, HEPES potassium and trisacetic acid
- various salts for example, surfactants, RNA polymerases (T7, T3, and SP6 RNA polymerases), chaperone proteins (DnaJ, DnaK, GroE). , GroEL, GroES, and HSP70), RNA (mRNA, tRNA, etc.), protease inhibitor, or (ribo) nuclease inhibitor.
- Ribosome display is a method for selecting a protein encoding a polypeptide having a specific function by forming an mRNA-ribosome-oligopeptide ternary complex in an in vitro translation system.
- the terminal modified sequence of the present invention has an arrest sequence at the 3 'end of the oligonucleotide library to be matured.
- a 5 ′ UTR sequence including a T7 promoter having an FLAG sequence added to the 3 ′ end and an SD sequence was also DNA-synthesized (SEQ ID NO: 2: gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatggactataaagatgacgatgacaaa).
- the partial sequence of geneIII (amino acid residues 220-326) of the M13 phage is the primer Myc-g3p (SEQ ID NO: 3: GAGCAGAAGCTGATCTCTGAGGAGGATCAAGATCTCGAGACGCGTTCTGTGGCTCGCTGTT using the M13KO7-derived phage genome as the template, and the primer g3p-SecMGATCGCTG PCR amplification by KOD Plus DNA Polymerase (Toyobo) (denaturation: 94 ° C., 15 seconds; annealing: 57 ° C., 30 seconds; extension: 68 ° C., 60 seconds; 25 cycles), followed by purification column (Qiagen) Purified.
- KOD Plus DNA Polymerase Toyobo
- Ice-cooled Wash buffer 50 mM Tris-OAc, pH 7.5, 150 mM NaCl, 50 mM Mg (OAc) 2 , 0.5 percent Tween 20, 10 ⁇ g / ml budding yeast (Saccharomyces cereviseae) total RNA (Sigma) 500 ⁇ L
- Blocking buffer 50 mM Tris-OAc, pH 7.5, 150 mM NaCl, 50 mM Mg (OAc) 2 , 0.5 percent Tween 20, 10 ⁇ g / ml budding yeast (Saccharomyces cereviseae) total 500 ⁇ L of RNA (Sigma), 5 percent SuperBlock (Pierce) was added.
- RT-PCR The recovered mRNA is converted to cDNA using a transcription high fidelity cDNA synthesis kit (Roche), and then PCR reaction using KOD plus DNA polymerase (denaturation: 94 ° C., 15 seconds; annealing: 57 ° C., 30 seconds; extension: 68 ° C., 60 seconds; 20 cycles).
- the primers used are shown below.
- Reverse transcription reverse primer Myc-R (SEQ ID NO: 7: CAGATCCTCCTCAGAGATCAGC)
- FIG. 1 shows a random oligonucleotide library (NNS sequence) before and after maturation.
- NPS sequence random oligonucleotide library
- Sequence number 1 Oligonucleotide n represents arbitrary bases. s represents guanine or cytosine. SEQ ID NO: 2: Oligonucleotide including T7 promoter, SD sequence and start codon SEQ ID NO: 3-8: Primer
- the method for producing a matured oligonucleotide library of the present invention can be used, for example, in the biochemical industry or the protein pharmaceutical industry.
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Abstract
Description
成熟化の対象とする9アミノ酸をコードするコドンを含むランダムオリゴヌクレオチドライブラリの5’側にFLAG配列,3’側にミック配列を付加した状態でDNA合成した(配列番号1:ATGGACTATAAAGATGACGATGACAAAnnsnnsnnsnnsnnsnnsnnsnnsnnsGAGCAGAAGCTGATCTCTGAGGAGGATCTG)。3‘末端にFLAG配列を付加したT7プロモーターおよびSD配列を含む5’ UTR配列もDNA合成した(配列番号2:gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatggactataaagatgacgatgacaaa)。M13ファージのgeneIIIの部分配列(アミノ酸残基220-326位)はM13KO7由来ファージゲノムを鋳型としてプライマーMyc-g3p(配列番号3:GAGCAGAAGCTGATCTCTGAGGAGGATCTGGAATATCAAGGCCAATCGTCTGAC)及び,プライマーg3p-SecMstop(配列番号4:CTCGAGTTATTCATTAGGTGAGGCGTTGAGGGCCAGCACGGATGCCTTGCGCCTGGCTTATCCAGACGGGCGTGCTGAATTTTGCGCCGGAAACGTCACCAATGAAAC)を用いてKOD Plus DNA Polymerase(東洋紡製)によってPCR増幅(変性:94℃,15秒;アニーリング:57℃,30秒;伸長:68℃,60秒;25サイクル)後,精製カラム(キアゲン社製)によって精製した。5’ UTR, ランダムオリゴヌクレオチドライブラリ,g3pの3種類のDNAをそれぞれ1 pmol,5’ プライマー(配列番号5:gaaattaatacgactcactatagggagaccacaacggtttccctctag) 10 pmol,SecMストップ配列(配列番号6:ggattagttattcattaggtgaggcgttgagg) 10 pmol,KOD Plus DNA ポリメラーゼ を含むPCR反応液を調製し,10サイクルのPCR反応(変性:94℃,15秒;アニーリング:57℃,30秒;伸長:68℃,60秒)を実行した。1%アガロースを用いた電気泳動によって,3遺伝子がつながったバンドを確認後,そのバンドを切り出した後カラム精製し(キアゲン製),最終的なランダム配列を含む成熟化対象オリゴヌクレオチドライブラリとした。
精製した成熟化対象オリゴヌクレオチドライブラリDNA 1μgを20μlのインビトロ転写キット(RibomaxTM Large Scale RNA Production System-T7, プロメガ社)によってmRNAとし,カラム精製した(RNeasy mini カラム,キアゲン社)。
予め5% SuperBlockで4℃において一晩ブロッキングしておいたFLAG M2 担体(50μLスラリー,シグマ社製)を500 μl Wash緩衝液でMicroSpin(登録商標)カラム(GEヘルスケア社製)を用いて2回洗浄後,回収したFLAG M2 担体に翻訳反応液を加え4℃で1時間ローテーションによって攪拌した。MicroSpin(登録商標)カラム(GEヘルスケア社製)によって上清を廃棄し,回収したFLAG M2 担体に1 mL のWash緩衝液を加え,4℃で5分ローテーションによって攪拌した。この操作を20回繰り返した後,100μlエルーション(Elution)緩衝液(50 mM Tris-OAc, pH7.5,150 mM NaCl,50 μg FLAGペプチド(シグマ社製))を回収したFLAG M2担体に加え,4℃で15分静置した。このようにして,複合体をFLAG M2担体から遊離させた。MicroSpin(登録商標)カラム(GEヘルスケア社製)によって上清を回収し,RNeasy Micro(キアゲン社製)によってmRNAを回収,精製した。
回収したmRNAはトランスクリプションハイフィデリティcDNA合成キット(ロッシュ社)によってcDNAとした後,KODプラスDNAポリメラーゼを用いてPCR反応(変性:94℃,15秒;アニーリング:57℃,30秒;伸長:68℃,60秒;20サイクル)を行った。なお,使用したプライマーを以下に示す。
Myc-R (配列番号7:CAGATCCTCCTCAGAGATCAGC)
選択前および選択後のDNA(100 ng)をrTaq DNAポリメラーゼ(東洋紡社製)によって3’末端にAを付加した。その後,TOPO TAクローニングキット(インビトロジェン社製)によってサブクローニングを行った。サブクローニングは,このキットの説明書に基づいて行った。形質転換後の大腸菌シングルコロニーをそれぞれ20コロニー3mlLB培地で培養し,増幅した大腸菌からプラスミドを回収し,塩基配列解析に使用した。
図1に,成熟化前後のランダムオリゴヌクレオチドライブラリ(NNS配列)を示す。
図1に示されるように,成熟化前の塩基配列解析の結果,ストップコドン(TAG)を含むオリゴヌクレオチドの出現頻度は40パーセント(8/20),塩基の欠失では5パーセント(1/20),塩基の挿入では0%(0/20)であり,最終的に完全なインフレームオリゴヌクレオチドの出現頻度は55%(11/20)であった。また,成熟化後においては,ストップコドン,塩基の欠失,挿入を含むものの出現頻度は0パーセント(0/20)であり,すべてのオリゴヌクレオチドがインフレームであることが確認された。この結果は,本法によるインフレームオリゴヌクレオチドの選択がほぼ完全に達成されていることを示しており,本法の有効性が証明された。
nは任意の塩基を表す。
sはグアニン又はシトシンを表す。
配列番号2:T7プロモーター,SD配列および開始コドンを含むオリゴヌクレオチド
配列番号3~8:プライマー
Claims (7)
- 成熟化対象オリゴヌクレオチドライブラリの5’末端にタグ配列を付加するとともに,前記成熟化対象オリゴヌクレオチドライブラリの3’末端にリボソームの翻訳伸長を停止させるアレスト配列を付加し,前記成熟化対象オリゴヌクレオチドライブラリの末端修飾物を得る工程と,
前記末端修飾配列物を転写させ,転写物を得る転写工程と,
前記転写物をインビトロで翻訳するインビトロ翻訳工程と,
を含み,
前記成熟化対象オリゴヌクレオチドライブラリがランダムオリゴヌクレオチドライブラリである,
成熟化オリゴヌクレオチドライブラリの製造方法。 - 前記ランダムオリゴヌクレオチドライブラリは,NNK配列,NNS配列又はNNY配列を含むオリゴヌクレオチドライブラリである請求項1に記載の方法。
- 前記リボソームの翻訳伸長を停止させるアレスト配列は,SecM配列である請求項1又は2に記載の方法。
- 前記タグ配列は,FLAG配列である請求項1~3のいずれか1項に記載の方法。
- 前記インビトロ翻訳工程は,
無細胞翻訳系を用いて前記転写物をインビトロで翻訳する工程である,
請求項1~4のいずれか1項に記載の方法。 - 前記インビトロ翻訳工程は,
大腸菌無細胞翻訳系を用いて前記転写物をインビトロで翻訳する工程である,
請求項1~5のいずれか1項に記載の方法。 - 前記インビトロ翻訳工程は,
再構成型無細胞翻訳系を用いて前記転写物をインビトロで翻訳する工程である,
請求項1~5のいずれか1項に記載の方法。
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US13/806,470 US9133452B2 (en) | 2010-06-23 | 2011-06-23 | High-speed maturation method for an oligonucleotide library for the purpose of preparing a protein library |
EP11798236.3A EP2586863B1 (en) | 2010-06-23 | 2011-06-23 | High-speed maturation method for an oligonucleotide library for the purpose of preparing a protein library |
JP2012521538A JP5850460B2 (ja) | 2010-06-23 | 2011-06-23 | 蛋白質ライブラリ調製を目的としたオリゴヌクレオチドライブラリの高速成熟化法 |
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JP2008271903A (ja) | 2007-05-02 | 2008-11-13 | Univ Of Tokyo | リボソームディスプレイ |
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EP2586863A4 (en) | 2013-12-25 |
JP5850460B2 (ja) | 2016-02-03 |
US20130143773A1 (en) | 2013-06-06 |
US9133452B2 (en) | 2015-09-15 |
JPWO2011162355A1 (ja) | 2013-08-22 |
EP2586863B1 (en) | 2017-05-03 |
EP2586863A1 (en) | 2013-05-01 |
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