JP2015023821A - Method for producing protein using secretion chaperone - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a protein with high efficiency using a transformant obtained by transforming a host with a vector comprising a polynucleotide encoding a protein.SOLUTION: The invention provides a method for increasing the amount of protein production by coexpressing a signal recognition particle and a protein added with a signal recognition particle-dependent signal peptide to the N-terminal side in a host; wherein the protein is human Fc binding protein, and the signal recognition particle is Ffh.

Description

  The present invention relates to a method for producing the protein with high efficiency using a transformant obtained by transforming a host with a vector containing a polynucleotide encoding the protein.

  Production of heterologous proteins using transformants (genetic recombinants) obtained by transforming a host with a vector containing a protein-encoding polynucleotide is widely used when producing useful proteins used in biopharmaceuticals, etc. Has been. However, when a protein having a disulfide bond in the molecule is to be expressed in the host, the protein may be reduced due to a reducing atmosphere in the host or the absence of disulfide isomerase in the host. A correct three-dimensional structure cannot be taken, and as a result, it may be produced as an inclusion body having no function as a protein.

In order to prevent this phenomenon, for example, in the production of a heterologous protein using genetically modified E. coli, an oligopeptide consisting of 15 to 30 amino acid residues called a signal peptide or leader peptide is added to the N-terminal side of the protein, There is known a method for secretory expression in the periplasmic region between the outer and inner membranes of Escherichia coli where an oxidative atmosphere and disulfide isomerase are present (Patent Document 1). In protein secretion in cells, the signal peptide has a function of binding a target protein to a secretory channel composed of a membrane protein such as SecA, SecY, SecE, or SecG on the inner membrane, and the target protein passes through the secretory channel. When transferred to the periplasmic region, the added signal peptide is separated and removed from the target protein by a protease called signal peptidase. The characteristics of the amino acid sequence constituting the signal peptide include the following three (Patent Document 1).
(I) The N-terminal region contains at least one basic amino acid such as arginine or lysine, and the signal peptide is bound to the cell membrane by ionic bond between the positive charge on the side chain of the basic amino acid and the negative charge on the cell membrane surface. Move in.
(Ii) The central region contains many hydrophobic amino acids such as alanine, leucine, isoleucine, and valine, and is involved in penetration into the cell membrane.
(Iii) In the C-terminal region, a specific amino acid cleaved by a signal peptidase after passing through the cell membrane exists as a recognition site, and the mature protein is released to the periplasmic region or outside the cell by being cleaved at the recognition site. Is done.

  To secrete and express the recombinant protein in the periplasmic region of the host, it is necessary to express the recombinant protein in a state where a signal peptide that promotes secretory expression to the periplasm is added to the N-terminal side of the protein. However, when the above method is used, there is a problem that productivity as an active protein is low. In addition, examples of improving the productivity by modifying the signal peptide have been reported (Patent Documents 1 to 3), but it was not sufficient for industrial production.

JP 2000-175686 A JP 2008-073044 A WO2008 / 032659

  An object of the present invention is to provide a method for producing the protein with high efficiency using a transformant obtained by transforming a host with a vector containing a polynucleotide encoding the protein.

  As a result of intensive studies to solve the above problems, the inventors of the present invention co-expressed a signal recognition particle and a protein having a signal recognition particle-dependent signal peptide added to the N-terminal side in the host. As a result, the present invention has been completed.

That is, the present invention includes the following inventions:
(A) The method for producing a protein, wherein a signal recognition particle and a protein having a signal recognition particle-dependent signal peptide added to the N-terminal side are co-expressed in a host.

  (B) The production method according to (A), wherein the protein is a human Fc-binding protein.

  (C) The production method according to (A) or (B), wherein the signal recognition particle is Ffh.

  (D) The production method according to (C), wherein the host is Escherichia coli.

  (E) The production method according to (D), wherein the signal recognition particle-dependent signal peptide is an oligopeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3.

  (F) The production method according to (D) or (E), wherein Ffh is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.

  Hereinafter, the present invention will be described in detail.

  In the production method of the present invention, a signal recognition particle (hereinafter referred to as SRP) -dependent peptide is added to the N-terminal side of a protein to be expressed in a host, and then the protein and SRP are co-expressed. It is characterized by manufacturing. SRP is one of the secretory chaperones and is a complex of protein and RNA. In eukaryotes, it is composed of 6 proteins (SRP9, SRP14, SRP19, SRP54, SRP68, SRP72) and 7S RNA, and in prokaryotes a protein with a molecular weight of 50,000 called Ffh, which is a homologue of SRP54, and 4.5S RNA (Saraogi et al., Traffic, 12 (5), 535-542, 2011). When a signal peptide portion of a protein is synthesized from an mRNA-ribosome complex, SRP is immediately bound to the protein and transferred to a secretory channel to form an mRNA-ribosome-synthetic protein-SRP-secretion channel complex. Is done. The protein passes through the secretory channel as it is synthesized and is folded into the correct conformation in the periplasmic region. The SRP and SRP-dependent signal peptide may be selected appropriately depending on the host in which the heterologous protein is to be expressed. For example, as a preferred example when the host is Escherichia coli, SRP is SEQ ID NO: 2 (UniProt No. P0AGD7), and the SRP-dependent signal peptide includes DsbA signal peptide (SEQ ID NO: 3, UniProt No. P0AEG4), TorT signal peptide, and TolB signal peptide.

  In order to co-express SRP and a protein having an SRP-dependent peptide added to the N-terminal side, a polynucleotide (polynucleotide A) encoding a protein having an SRP-dependent peptide added to the N-terminal side and a polymorphism encoding SRP A nucleotide (polynucleotide B) may be inserted into the same expression vector and co-expressed by culturing a transformant obtained by transforming the host with the vector, or polynucleotide A and polynucleotide B may be respectively expressed. It may be inserted into different expression vectors and co-expressed by culturing a transformant obtained by transforming a host with the two expression vectors. When the host is Escherichia coli, examples of expression vectors for inserting polynucleotide A and polynucleotide B include pTrc and pUC. Inserting polynucleotide A when inserting polynucleotide A and polynucleotide B into different expression vectors. An example of an expression vector to be inserted is pTrc, and an example of an expression vector for inserting polynucleotide B is pACYC.

  The protein produced by the production method of the present invention is not particularly limited, and examples thereof include human-derived proteins such as insulin, interferon, interleukin, antibody, erythropoietin, growth hormone, and their receptor proteins. The protein produced by the production method of the present invention may be a complete protein, a protein composed only of a part important for the function of the protein, or an amino acid constituting the protein. One or more of these may be deleted and / or inserted and / or substituted. Hereinafter, human Fc-binding proteins among proteins that can be produced by the production method of the present invention will be described in detail.

In this specification, human Fc-binding protein refers to the extracellular region of human FcγRI (specifically, in the case of natural human FcγRI, from the 16th glutamine to the 292nd histidine in the amino acid sequence described in SEQ ID NO: 1). Region) (refer to FIG. 1). However, it does not necessarily have to be the entire region of the human FcγRI extracellular region. Of the proteins (polypeptides) constituting the human FcγRI extracellular region, it expresses the original function of binding to at least the Fc region of an antibody (immunoglobulin). It only needs to contain the polypeptide of the region to be obtained. As an example of the human Fc binding protein,
(I) a protein comprising an amino acid residue from at least the 16th glutamine to the 289th valine in the amino acid sequence set forth in SEQ ID NO: 1,
(Ii) including at least the amino acid residue from the 16th glutamine to the 289th valine in the amino acid sequence of SEQ ID NO: 1, and replacing one or more of the amino acid residues with another amino acid residue , Inserted or deleted proteins.

Specific examples of the above (ii) include the Fc binding protein disclosed in JP2011-206046 and the following amino acid residues in the 34th to 307th amino acid residues of the amino acid sequence set forth in SEQ ID NO: 31 ( Examples thereof include Fc-binding proteins (Japanese Patent Application No. 2012-270375) in which at least one of substitutions among 1) to (42) has occurred.
(1) 37th threonine of SEQ ID NO: 31 is replaced with isoleucine (2) 38th proline of SEQ ID NO: 31 is replaced with serine (3) 53rd leucine of SEQ ID NO: 31 is replaced with glutamine (4) SEQ ID NO: 31: 62nd glutamic acid is replaced with valine (5) 63rd valine of SEQ ID NO: 31 is replaced with alanine or glutamic acid (6) 66th leucine of SEQ ID NO: 31 is replaced with glutamine or proline (7) SEQ ID NO: 31 67 of serine is replaced with proline (8) 69th alanine of SEQ ID NO: 31 is replaced with valine or threonine (9) 71st serine of SEQ ID NO: 31 is replaced with threonine or leucine (10) 78th aspartic acid is replaced by glutamic acid (11) 81st isoleucine of SEQ ID NO: 31 is (12) The 84th serine of SEQ ID NO: 31 is replaced with threonine (13) The 88th phenylalanine of SEQ ID NO: 31 is replaced with tyrosine (14) The 95th glutamic acid of SEQ ID NO: 31 is replaced with aspartic acid ( 15) 119th histidine of SEQ ID NO: 31 replaced with glutamine (16) 127th valine of SEQ ID NO: 31 replaced with alanine (17) 146th arginine of SEQ ID NO: 31 replaced with lysine (18) SEQ ID NO: 31 The aspartic acid at position 147 was replaced with asparagine (19) The histidine at position 151 in SEQ ID NO: 31 was replaced with tyrosine (20) The threonine at position 178 in SEQ ID NO: 31 was replaced with alanine (21) The 191st position in SEQ ID NO: 31 Arginine is replaced with lysine (22) The 199th threonine of SEQ ID NO: 31 is (23) 200th leucine of SEQ ID NO: 31 replaced with methionine (24) 213th threonine of SEQ ID NO: 31 replaced with alanine (25) 216th valine of SEQ ID NO: 31 replaced with alanine (26 ) The 221st leucine of SEQ ID NO: 31 was replaced with arginine (27) The 229th serine of SEQ ID NO: 31 was replaced with asparagine (28) The 236th isoleucine of SEQ ID NO: 31 was replaced with lysine (29) 244th tyrosine replaced with histidine (30) 253rd threonine of SEQ ID NO: 31 replaced with alanine (31) 290th arginine of SEQ ID NO: 31 replaced with glutamine (32) 293rd lysine of SEQ ID NO: 31 replaced Substitute for asparagine (33) The 297th lysine of SEQ ID NO: 31 was substituted for glutamic acid. Substitution (34) The 306th proline of SEQ ID NO: 31 is replaced with threonine (35) The 34th glutamine of SEQ ID NO: 31 is substituted with arginine (36) The 45th glutamine of SEQ ID NO: 31 is substituted with lysine (37) The 82nd glutamine of No. 31 is replaced with proline (38) The 177th asparagine of SEQ ID NO: 31 is replaced with aspartic acid (39) The 213th threonine of SEQ ID NO: 31 is substituted with serine (40) 242 of SEQ ID NO: 31 The 41st glutamine is replaced with arginine (41) The 253rd threonine of SEQ ID NO: 31 is replaced with serine (42) The 271st glutamic acid of SEQ ID NO: 31 is replaced with aspartic acid as a host used in the production method of the present invention. Cells, animal cells represented by CHO cells, Bacillus (Brevibacillus bacteria, Examples include bacteria represented by Escherichia coli, bacteria represented by Escherichia coli, yeasts represented by the genus Saccharomyces, Pichia and Schizosaccharomyces, and filamentous fungi represented by Neisseria gonorrhoeae. It is preferable to use E. coli which is easy to handle as a host. When the host is E. coli and the protein is a human Fc binding protein, the human Fc binding protein and SRP are co-expressed by culturing the genetically modified E. coli by the method disclosed in JP2012-034591A. Just do it.

In the production method of the present invention, in order to recover a protein expressed from a host culture solution, it may be appropriately selected depending on the form of expression. For example, when the expressed protein is expressed in the periplasmic region of the host cell, the host cell obtained by centrifuging the culture medium is suspended in an appropriate buffer, and after cell disruption (physical disruption, disruption with drugs, etc.) The cell-free extract containing the expressed protein can be obtained by removing the crushing residue by centrifugation. If the expressed protein leaks from the periplasmic region of the host cell to the culture supernatant, the culture solution is centrifuged. The protein expressed from the culture supernatant obtained in this manner may be recovered. When the host cells are disrupted with a drug, for example, 150 mM NaCl, 1 mM EDTA, 6 mM MgSO ₄ , 250 U / L Benzonase (trade name), 0.3 g / L Lysozyme, 0.4% Triton X-100, 0. Commercial extraction such as 50 mM Tris-HCl (pH 8.0) (Japanese Patent Application No. 2012-129732) containing 5% CTAB (hexadecyltrimethylammonium bromide) and 50 mM CaCl ₂ and BugBuster Protein extraction kit (manufactured by Novagen) It is good to crush using a reagent.

  The solution containing the recovered protein can be purified and isolated by cation exchange chromatography, hydrophobic chromatography, or the like. The carrier for cation exchange chromatography is obtained by binding a cation exchange group such as carboxymethyl group or sulfopropyl group to the carrier. As an example, TOYOPEARL CM-650, TOYOPEARL SP-650 (above, manufactured by Tosoh Corporation), CM Sepharose FastFlow (manufactured by GE Healthcare) can be mentioned. The carrier for hydrophobic chromatography is obtained by binding a hydrophobic functional group such as an ether group, a phenyl group or a butyl group to the carrier. Manufactured by Tosoh Corporation). When purification is performed using various chromatographic carriers, an appropriate open column or the like may be filled with the amount of carrier determined by the amount of Apply solution introduced or the protein adsorption performance of the carrier. The chromatographic carrier is equilibrated in advance with an appropriate buffer (Tris / Tris hydrochloride buffer, glycine / sodium hydroxide buffer, phosphate buffer, etc.) before introducing the apply solution. . The protein-containing solution recovered by the above-described method is introduced into an equilibrated chromatographic support so that the protein is adsorbed onto the support and washed with the same buffer as that used for equilibration. Thereafter, a purified solution containing the protein can be obtained by eluting the adsorbed protein using an elution buffer.

  The present invention is characterized in that the protein is produced by co-expressing a signal recognition particle (SRP) and a protein having an SRP-dependent signal peptide added to the N-terminal side in a host. According to the production method of the present invention, it is possible to greatly improve the production amount of recombinant protein, and the production efficiency of industrially useful recombinant protein can be greatly improved.

The figure which shows the structure of natural type human FcγRI.

  Hereinafter, the present invention will be described in more detail using Examples and Comparative Examples when a human Fc-binding protein is used as a protein, but the present invention is not limited to the above examples.

Example 1 Production of Signal Recognizing Particle (SRP) Protein Expression Vector (1) Escherichia coli W3110 strain was inoculated on LB plate medium and cultured at 30 ° C. overnight.
(2) An E. coli colony of (1) as a template, and an oligonucleotide consisting of the sequence described in SEQ ID NO: 4 (5′-gaattcgtgcacgataatagtgttattattatta-3 ′) and SEQ ID NO: 5 (5′-gaattcctgcagttagcgaccagagggaa-3 ′) as a PCR primer PCR was performed. After preparing the reaction solution shown in Table 1, the PCR was heated at 98 ° C. for 5 minutes, the first step for 10 seconds at 98 ° C., the second step for 5 seconds at 55 ° C., and 90 seconds at 72 ° C. After repeating the reaction which made the 3rd step of 1 cycle 30 cycles, it carried out by heating at 72 degreeC for 7 minutes. The obtained PCR product was purified using a Qiagen PCR purification kit to obtain a polynucleotide (about 1.5 kbp) encoding Ffh (SEQ ID NO: 2) which is a protein component of SRP.

（３）得られたＦｆｈをコードするポリヌクレオチドを制限酵素Ａｌｗ４４ＩおよびＰｓｔＩで消化し、キアゲン社製ＰＣＲ精製キットを用いて精製した。
（４）市販ベクターｐＡＣＹＣ１７７（ニッポンジーン社）を制限酵素Ａｌｗ４４ＩおよびＰｓｔＩで消化し、キアゲン社製ＰＣＲ精製キットを用いて精製した。得られた制限酵素消化ベクターは、タカラバイオ社製アルカリフォスファターゼを用いて５０℃で３０分処理後、キアゲン社製ＰＣＲ精製キットを用いて精製することで、約３．５ｋｂｐの一本鎖ポリヌクレオチドを得た。
（５）（２）と（３）で得たポリヌクレオチドを混合し、ＤＮＡ Ｌｉｇａｔｉｏｎ ｋｉｔ（タカラバイオ社製）を用いてライゲーションし、このライゲーション産物を用いて大腸菌ＪＭ１０９株（タカラバイオ社製）を形質転換した。
（６）得られた形質転換体を５０μｇ／ｍＬのカナマイシン（和光純薬社製）を含むＬＢ培地にて培養後、ＱＩＡｐｒｅｐ Ｓｐｉｎ Ｍｉｎｉｐｒｅｐ ｋｉｔ（キアゲン社製）を用いて、発現ベクターｐＡＣＹＣＳＲＰを抽出した。
（７）（６）で作製した発現ベクターｐＡＣＹＣＳＲＰのうち、Ｆｆｈおよびその周辺の領域について、チェーンターミネータ法に基づくＢｉｇ Ｄｙｅ Ｔｅｒｍｉｎａｔｏｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ ＦＳ ｒｅａｄ Ｒｅａｃｔｉｏｎ ｋｉｔ（ライフテクノロジー社製）を用いてサイクルシークエンス反応に供し、全自動ＤＮＡシークエンサーＡＢＩ Ｐｒｉｓｍ ３７００ ＤＮＡ ａｎａｌｙｚｅｒ（ライフテクノロジー社製）にてヌクレオチド配列を解析した。なお当該解析の際、配列番号７（５’−ｔｃｃｃｔｔｔｔｔｔｇｃｇｇｃａｔｔｔｔｇｃｃｔｔｃｃｔｇｔｔｔｔｔｇｃｔｃａｃ−３’）または配列番号８（５’−ｃｇｃｃｔｃｃａｔｃｃａｇｔｃｔａｔｔａａｔｔｇｔｔｇｃｃｇｇｇａａｇｃｔａｇａ−３’）に記載の配列からなるオリゴヌクレオチドをシークエンス用プライマーとして使用した。解析の結果設計通りであることを確認した。Ｆｆｈ（配列番号２）をコードするポリヌクレオチドの配列を配列番号６に示す。

(3) The obtained polynucleotide encoding Ffh was digested with restriction enzymes Alw44I and PstI and purified using a Qiagen PCR purification kit.
(4) A commercially available vector pACYC177 (Nippon Gene) was digested with restriction enzymes Alw44I and PstI and purified using a PCR purification kit manufactured by Qiagen. The obtained restriction enzyme digestion vector was treated with alkaline phosphatase manufactured by Takara Bio Co., Ltd. for 30 minutes at 50 ° C., and then purified using a PCR purification kit manufactured by Qiagen Co. to obtain a single-stranded polynucleotide of about 3.5 kbp. Got.
(5) The polynucleotides obtained in (2) and (3) are mixed and ligated using a DNA ligation kit (manufactured by Takara Bio Inc.), and Escherichia coli JM109 strain (manufactured by Takara Bio Inc.) is used using this ligation product. Transformed.
(6) After culturing the obtained transformant in an LB medium containing 50 μg / mL kanamycin (manufactured by Wako Pure Chemical Industries, Ltd.), the expression vector pACYCSRP was extracted using QIAprep Spin Miniprep kit (manufactured by Qiagen). .
(7) Of the expression vector pACYCSRP prepared in (6), Ffh and the surrounding region are subjected to a cycle sequence reaction using the Big Dye Terminator Cycle Sequencing FS read Reaction kit (manufactured by Life Technologies) based on the chain terminator method. The nucleotide sequence was analyzed using a fully automatic DNA sequencer ABI Prism 3700 DNA analyzer (manufactured by Life Technology). During the analysis, SEQ ID NO: 7 (5′-tccccttttgcggcatattttgccttcctgtttttgtctcac-3 ′) or SEQ ID NO: 8 (5′-cgccttccattccattctattattattgtttgccgggaagctaggatagctaga-3 ′) was used as a primer for the sequence. As a result of analysis, it was confirmed that it was as designed. The sequence of the polynucleotide encoding Ffh (SEQ ID NO: 2) is shown in SEQ ID NO: 6.

Comparative Example 1 Preparation of SecB Expression Vector For comparison, a vector capable of expressing SecB, a secretory chaperone different from SRP, was prepared. SecB is thought to bind to the protein produced in the cytoplasm and prevent protein aggregation and keep it unfolded until it passes through the secretion channel (Collier et al., Cell, 53 (2), 273-283). 1988). Proteins such as PelB, OmpA, PhoA, and pIII are secreted through SecB.

  Example 1 (1) to (6) except that oligonucleotides having the sequences described in SEQ ID NO: 9 (5′-gaattcgtgcacgataatagtcagaaaaac-3 ′) and SEQ ID NO: 10 (5′-gaattcctgcagtcaggcatcctgtgg-3 ′) were used as primers. The expression vector pACYCecB was obtained by inserting the approximately 470 bp polynucleotide encoding SecB (SEQ ID NO: 11) into the commercial vector pACYC177. In addition, the same operation as in Example 1 (7) was performed, and it was confirmed that the sequence of the polynucleotide encoding SecB inserted into the vector was as designed. The sequence of the polynucleotide encoding SecB is shown in SEQ ID NO: 12.

Example 2 Construction of pTrcdsb Vector An oligonucleotide encoding a DsbA signal peptide (amino acid sequence: MKKIWLALAGLVLAFSASA, SEQ ID NO: 3), which is one of SRP-dependent signal peptides, was expressed by the following method using the expression vector pTrc99a (GE Healthcare). The expression vector pTrcdsb was prepared by inserting the product into the product.
(1) SEQ ID NO: 13 (5'-catgaaaaaaatctggctggcgctggcgggtctggtgctggcgtttagcgcgagcgcgaaaatcgaagaagc-3 ') and SEQ ID NO: 14 (5'-catggcttcttcgattttcgcgctcgcgctaaacgccagcaccagacccgccagcgccagccagattttttt-3' The oligonucleotides were mixed in equal amounts consisting of the sequences described), after heating for 5 minutes at 95 ° C., Double-stranded oligonucleotide DsbAp1 was prepared by lowering the temperature every 1 ° C. over 1 minute until reaching 15 ° C. DsbAp1 was kept at 15 ° C. until use.
(2) DsbAp1 prepared in (1) was ligated to the expression vector pTrc99a previously digested with the restriction enzyme NcoI using DNA Ligation kit (Takara Bio Inc.), and E. coli JM109 strain (Takara) was used with this ligation product. Bio).
(3) After culturing the obtained transformant in an LB medium containing 100 μg / mL carbenicillin sodium (manufactured by Wako Pure Chemical Industries, Ltd.), the expression vector pTrcdsb is extracted using QIAprep Spin Miniprep kit (manufactured by Qiagen). did.
(5) Big Dye Terminator Cycle Sequencing FS read Reaction kit (Life Technologies) based on the chain terminator method for the polynucleotide encoding DsbA signal peptide and its peripheral region in the expression vector pTrcdsb prepared in (4) The nucleotide sequence was analyzed with a fully automatic DNA sequencer ABI Prism 3700 DNA analyzer (manufactured by Life Technology Co., Ltd.). In the analysis, an oligonucleotide consisting of the sequence shown in SEQ ID NO: 15 (5′-tgtgggtgggtgtgtcagg-3 ′) or SEQ ID NO: 16 (5′-tcggcatgggggtcagtgt-3 ′) was used as a sequencing primer. As a result of analysis, it was confirmed that it was as designed.

Example 3 Preparation of human Fc binding protein (FcRm68) expression vector pTrcdsbFcRm68-CG Human Fc binding protein (amino acid sequence: CG) added to the expression vector pTrcdsb prepared in Example 2 on the C-terminal side (amino acid sequence: CG) An FcRm68-CG expression vector pTrcdsbFcRm68-CG was constructed by inserting a polynucleotide encoding FcRm68-CG). The human Fc binding protein FcRm68 (SEQ ID NO: 18) is a human Fc binding protein FcRm60c consisting of the region from the 34th glutamine to the 307th aspartic acid in the amino acid sequence shown in SEQ ID NO: 17 No. 206046)
37th threonine of SEQ ID NO: 17 is isoleucine,
The 63rd valine of SEQ ID NO: 17 is glutamic acid,
The 69th alanine of SEQ ID NO: 17 is valine,
The 71st serine of SEQ ID NO: 17 is leucine,
84th serine of SEQ ID NO: 17 is threonine,
95th glutamic acid of SEQ ID NO: 17 is aspartic acid,
The 292nd proline of SEQ ID NO: 17 is lysine,
The 293rd glutamic acid of SEQ ID NO: 17 is lysine,
The 297th glutamine of SEQ ID NO: 17 is lysine,
The 301st histidine of SEQ ID NO: 17 is lysine,
The 304th proline of SEQ ID NO: 17 is lysine,
Each is a human Fc-binding protein with amino acid substitution. The amino acid sequence of the human Fc binding protein FcRm60c (SEQ ID NO: 17) corresponds to the region from the 16th glutamine to the 289th valine in the amino acid sequence of the human Fc receptor FcγRI described in SEQ ID NO: 1. In 60 amino acid substitutions occurred in JP 2011-206046.
(1) SEQ ID NO: 20 (5'-catgccatgggacaattaggccataggctgtgtattactctgcaccgtgtgtgtgtgtgtgtggtgtgtggtgtgtg PCR was performed using the oligonucleotide consisting of the sequence as a PCR primer. In PCR, after preparing a reaction solution having the composition shown in Table 1, the reaction solution was subjected to a first step at 98 ° C. for 10 seconds, a second step at 50 ° C. for 5 seconds, and a third step at 72 ° C. for 1 minute. The reaction for 1 cycle was repeated 30 times. By purifying the obtained PCR product, a polynucleotide encoding the human Fc binding protein FcRm68-CG was obtained.
(2) The obtained polynucleotide encoding FcRm68-CG was digested with restriction enzymes NcoI and HindIII, and then ligated to the expression vector pTrcdsb prepared in Example 2 previously digested with restriction enzymes NcoI and HindIII. Escherichia coli W3110 strain was used to transform it.
(3) An expression vector containing a polynucleotide encoding FcRm68-CG using QIAprep Spin Miniprep kit (manufactured by Qiagen) after culturing the obtained transformant in an LB medium supplemented with 100 μg / mL carbenicillin sodium. pTrcdsbFcRm68-CG was extracted.
(4) Among the expression vector pTrcdsbFcRm68-CG produced in (3), the nucleotide sequence of the polynucleotide encoding FcRm68 and the surrounding region were analyzed in the same manner as in Example 2 (5).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcdsbFcRm68-CG is shown in SEQ ID NO: 22, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 23, respectively. In SEQ ID NO: 22, the amino acid sequence of the DsbA signal peptide (SEQ ID NO: 3) is from the first methionine to the 19th alanine, and the amino acid sequence of the human Fc binding protein FcRm68 is from the 27th glutamine to the 300th aspartic acid. (SEQ ID NO: 18) The amino acid sequence of the cysteine tag is from the 301st cysteine to the 302nd glycine. In SEQ ID NO: 22, the amino acid sequence of FcRm68 (the region from the 27th glutamine to the 300th aspartic acid) is the amino acid sequence of the human Fc receptor FcγRI described in SEQ ID NO: 1 from the 16th glutamine to the 289th position. It corresponds to the area up to valine.

Comparative Example 2 Construction of Human Fc Binding Protein (FcRm68) Expression Vector pTrcpelFcRm68-CG For comparison, a PelB signal peptide (amino acid sequence: MKYLLPTAAAGLLLLAAQPAMA, SEQ ID NO: 24), which is one of SecB-dependent signal peptides, A vector pTrcpelFcRm68-CG capable of expressing a polypeptide added to the N-terminal side of the sex protein FcRm68-CG was prepared.
(1) as an oligonucleotide, except for using SEQ ID NO: 25 (5'-catgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggc-3 ') and SEQ ID NO: 26 (5'-catggccatcgccggctgggcagcgaggagcagcagaccagcagcagcggtcggcagcaggtattt-3') consisting of a sequence according to oligonucleotides Example 2 By performing the same operation, an expression vector pTrcpel in which a polynucleotide encoding a PelB signal peptide was inserted into pTrc99a was prepared.
(2) An expression vector pTrcpelFcRm68-CG was prepared by performing the same operation as in Example 3 (1) to (3) except that the expression vector pTrcpel prepared in (1) was used as the expression vector.
(3) The nucleotide sequence of the expression vector pTrcpelFcRm68-CG was analyzed in the same manner as in Example 2 (5).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcpelFcRm68-CG is shown in SEQ ID NO: 27, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 28, respectively. In SEQ ID NO: 27, the amino acid sequence of the PelB signal peptide (SEQ ID NO: 24) is from the first methionine to the 22nd alanine, and the amino acid sequence of the human Fc binding protein FcRm68 is from the 25th glutamine to the 298th aspartic acid. (SEQ ID NO: 18) The amino acid sequence of the cysteine tag is from the 299th cysteine to the 300th glycine. In SEQ ID NO: 27, the amino acid sequence of FcRm68 (region from the 25th glutamine to the 298th aspartic acid) is the amino acid sequence of the human Fc receptor FcγRI described in SEQ ID NO: 1 from the 16th glutamine to the 289th position. It corresponds to the area up to valine.

Example 4 Transformation with pACYCSRP and pTrcdsbFcRm68-CG and Evaluation of Human Fc Binding Protein Productivity (1) Using the Ffh expression vector pACYCSRP prepared in Example 1 and the FcRm68-CG expression vector pTrcdsbFcRm68-CG prepared in Example 3 E. coli strain W3110 was transformed, and the transformant was isolated on an LB plate medium supplemented with 50 μg / mL kanamycin and 100 μg / mL sodium ampicillin.
(2) The inoculated colonies were inoculated into 20 mL of LB medium supplemented with 50 μg / mL kanamycin and 100 μg / mL ampicillin sodium, and then cultured with shaking at 30 ° C. for 3 hours in a 100 mL shake flask. After cooling, IPTG was added to a concentration of 0.05 mM, and further cultured with shaking at 20 ° C. for 2 days.
(3) 1 mL of the culture solution of (2) was centrifuged to separate the culture supernatant and the bacterial cells. The collected cells were lysed by adding 1 mL of Bugbuster solution (manufactured by Novagen) and permeating for 20 minutes, and centrifuged again to remove the residue to obtain a cell extract.
(4) The amount of human Fc-binding protein in the culture supernatant and the bacterial cell extract obtained in (3) was measured by the ELISA method shown below.
(4-1) A gamma globulin preparation which is a human antibody (manufactured by Chemical and Serum Therapy Research Laboratories) was fixed to a well of a 96-well microplate at a concentration of 1 μg / well (18 hours at 4 ° C.), and after the immobilization was completed , Washed with a wash buffer (10 mM Tris-HCl buffer (pH 8.0) containing 0.05% (w / v) Tween 20 (trade name) and 150 mM NaCl) and 1.0% BSA ( Blocking was performed by Sigma-Aldrich).
(4-2) After washing with the washing buffer, the acid treatment solution was appropriately diluted with 50 mM Tris-HCl buffer (pH 8.0) containing 1.0 M NaCl and reacted with immobilized gamma globulin. (1 hour at 30 ° C.).
(4-3) After completion of the reaction, the plate was washed again with a washing buffer, and Anti-hFcγR1 / CD64 antibody reagent (R & D Systems) was added (1 hour at 30 ° C.).
(4-4) After the reaction was completed, the plate was washed again with the washing buffer, and Horse radish Peroxidase (HRP) -labeled Goat anti-Mouse IgG-h + IHRP antibody reagent (BETYL) was added.
(4-5) After reacting at 30 ° C. for 1 hour, the plate was washed with the washing buffer, TMB Peroxidase Substrate (manufactured by KPL) was added, and the absorbance at 450 nm was measured.

  As a result of the quantification, it was confirmed that 4.9 mL / L in the culture supernatant and 2.6 mg / L in the bacterial cell extract were produced, producing a total of 7.5 mg / L of human Fc-binding protein.

Comparative Example 3 Transformation with pTrcdsbFcRm68-CG alone and evaluation of human Fc binding protein productivity (1) E. coli W3110 strain was transformed with only the FcRm68-CG expression vector pTrcdsbFcRm68-CG prepared in Example 3, and 100 μg / mL Transformants were isolated on LB plate medium supplemented with sodium ampicillin.
(2) The inoculated colonies were inoculated into 20 mL of LB medium supplemented with 100 μg / mL ampicillin sodium, cultured in a 100 mL shake flask at 30 ° C. for 3 hours, cooled to 20 ° C., and 0.05 mM. Then, IPTG was added and further cultured with shaking at 20 ° C. for 2 days.
(3) Human Fc-binding protein productivity was evaluated by performing the same operations as in Examples 4 (3) to (4).

  As a result of quantification, it was confirmed that 2.2 mL / L of the culture supernatant and 3.6 mg / L of the bacterial cell extract were produced, producing a total of 5.8 mg / L of human Fc-binding protein. Although the expression level of the human Fc binding protein in the bacterial cell extract was higher than that in Example 4, the expression level of the human Fc binding protein in the culture supernatant was low (less than half), and the total expression level Was also low. That is, by co-expressing a signal recognition particle (SRP) and a target protein to which an SRP-dependent signal peptide is added on the N-terminal side in a host, the productivity of the target protein (especially to the culture supernatant) is improved. I understand that.

Comparative Example 4 Transformation with pACYCsecB and pTrcdsbFcRm68-CG and Evaluation of Human Fc Binding Protein Productivity As a vector for transforming Escherichia coli W3110 strain, the SecB expression vector pACYCsecB prepared in Comparative Example 1 and Example 2 were used. Human Fc-binding protein productivity was evaluated in the same manner as in Example 4 except that the FcRm68-CG expression vector pTrcdsbFcRm68-CG was used.

  As a result of quantification, the production amount of human Fc-binding protein was 2.6 mL / L in the culture supernatant, 2.2 mg / L in the bacterial cell extract, and a total of 4.8 mg / L. It was low compared with. That is, it can be seen that co-expression of a target protein to which an SRP-dependent signal peptide is added on the N-terminal side and a secretory chaperone (SecB) other than SRP does not contribute to an improvement in the productivity of the target protein.

Comparative Example 5 Transformation with pACYCSRP and pTrcpelFcRm68-CG and Evaluation of Human Fc Binding Protein Productivity As a vector for transforming Escherichia coli W3110 strain, the Ffh expression vector pACYCSRP prepared in Example 1 and Comparative Example 2 were used. Except for using the expression vector pTrcpelFcRm68-CG, the same operation as in Example 4 was performed to evaluate human Fc binding protein productivity.

  As a result of quantification, the production amount of human Fc-binding protein was 0.38 mL / L in the culture supernatant, 2.6 mg / L in the bacterial cell extract, and a total of 2.9 mg / L, both in Example 4. It was low compared with. That is, it can be seen that co-expression of SRP and a target protein to which a SecB-dependent signal peptide is added on the N-terminal side does not contribute to an improvement in the productivity of the target protein.

Comparative Example 6 Transformation with pTrcpelFcRm68-CG alone and evaluation of human Fc-binding protein productivity Other than using only the expression vector pTrcpelFcRm68-CG prepared in Comparative Example 2 as a vector for transforming E. coli W3110 strain, The same operation as in Comparative Example 3 was performed to evaluate human Fc binding protein productivity.

  As a result of quantification, human Fc-binding protein was produced at 2.3 mg / L only in the bacterial cell extract, and was not expressed in the culture supernatant.

Comparative Example 7 Transformation with pACYCsecB and pTrcpelFcRm68-CG and Evaluation of Human Fc Binding Protein Productivity As a vector for transforming Escherichia coli W3110 strain, the SecB expression vector pACYCSecB prepared in Comparative Example 1 and Comparative Example 2 were used. Except for using only the expression vector pTrcpelFcRm68-CG, the same operation as in Comparative Example 3 was performed to evaluate human Fc-binding protein productivity.

  As a result of quantification, the Fc-binding protein was produced at 2.9 mg / L only in the bacterial cell extract, and was not expressed in the culture supernatant. That is, it can be seen that co-expression of SecB and a target protein with a SecB-dependent signal peptide added to the N-terminal side in the host does not contribute to an improvement in the productivity of the target protein.

Example 5 Preparation of SRP / Fc binding protein tandem expression vector, transformation with the vector and evaluation of human Fc binding protein productivity In Example 4, a protein in which SRP and SRP-dependent signal peptide were added to the N-terminal side When a host is transformed with a vector containing a polynucleotide encoding SRP and a vector containing a polynucleotide encoding a protein with an SRP-dependent signal peptide added to the N-terminal side, In this example, a host is transformed with one vector containing a polynucleotide encoding an SRP and a polynucleotide encoding an SRP-dependent signal peptide added to the N-terminal side, and the productivity of the protein is determined. Evaluated.
(1) Using the pACYCSRP prepared in Example 1 as a template, an oligonucleotide consisting of the sequence of SEQ ID NO: 29 (5′-agttggaagctttagtttgtatattatta-3 ′) and SEQ ID NO: 30 (5′-ccattgaagcttttagcgaccaggaga-3 ′) as a primer Otherwise, PCR was performed in the same manner as in Example 1 (2) to obtain a polynucleotide encoding Ffh.
(2) FcRm68-CG expression vector pTrcdsbFcRm68-CG prepared in Example 3 was digested with HindIII, purified using QIAprep Spin Miniprep kit (Qiagen), and then 30 minutes at 50 ° C. with Takara Bio alkaline phosphatase. The vector was purified using a Qiagen PCR purification kit.
(3) The polynucleotide obtained in (1) was digested with the restriction enzyme HindIII, ligated to the vector obtained in (2), and Escherichia coli BL21 strain (Takara Bio Inc.) was transformed with this ligation product. .
(4) The obtained transformant is cultured in an LB medium supplemented with 100 μg / mL ampicillin sodium, and then encoded with a polynucleotide encoding FcRm68-CG and SRP using QIAprep Spin Miniprep kit (Qiagen). The expression vector pTrcdsbFcRm68-CG-SRP containing the polynucleotide to be extracted was extracted.
(5) The expression vector pTrcdsbFcRm68-CG-SRP obtained in (4) was subjected to sequence analysis in the same manner as in Example 2 (5) to confirm that the sequence was as designed.
(6) The transformant obtained in (3) was subjected to the same operation as in Example 4 to confirm the productivity of human Fc binding protein.

  As a result of the quantification, it was confirmed that the human Fc-binding protein was produced in a total amount of 6.9 mg / L, 4.7 mg / L in the culture supernatant and 2.2 mg / L in the bacterial cell extract. That is, using a transformant obtained by transforming a host with one vector containing a polynucleotide encoding a protein to which a polynucleotide encoding SRP and an SRP-dependent signal peptide are added at the N-terminal side, It can be seen that the productivity of the protein can also be improved by co-expressing the protein with SRP-dependent signal peptide added to the N-terminal side.

  Table 2 summarizes the results of Examples 4 and 5 and Comparative Examples 3 to 7.

Claims (6)

A method for producing the protein, comprising co-expressing a signal recognition particle and a protein having a signal recognition particle-dependent signal peptide added to the N-terminal side in a host. The production method according to claim 1, wherein the protein is a human Fc-binding protein. The production method according to claim 1 or 2, wherein the signal recognition particles are Ffh. The production method according to claim 3, wherein the host is Escherichia coli. The production method according to claim 4, wherein the signal recognition particle-dependent signal peptide is an oligopeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3. The production method according to claim 4 or 5, wherein Ffh is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.

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