JP2014223064A - Signal peptide and method for producing proteins using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a signal peptide which can efficiently transfer recombinant protein expressed in a host cell to a periplasmic region, and to provide a method for producing the protein using the peptide concerned.SOLUTION: The subject is solved by a signal peptide, among natural PelB signal peptides, inserted a basic amino acid in any of the N terminal region and by producing the protein using the peptide as a signal peptide for protein secretion of a periplasm.

Description

  The present invention relates to a novel signal peptide and a protein production method using the same.

Proteins that are synthesized in the cell and remain in the cell as they are, or those that are released as secreted proteins to the outside of the cell are known. In recombinant protein production using Escherichia coli or the like as a host, a method of secreting and expressing the protein in the periplasmic region between the inner membrane and outer membrane is known (Patent Document 1). When a recombinant protein is transferred from the cell to the periplasm region, the function of an oligopeptide called a signal peptide is important (Patent Document 1). 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.

  In order to cause a recombinant protein to be secreted and expressed in the host periplasm, it is necessary to express the recombinant protein in a state in which 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 signal peptide capable of efficiently transferring a recombinant protein expressed in a host cell to the periplasmic region, and a method for producing the protein using the peptide.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have improved the amount of recombinant protein produced by using an oligopeptide having a basic amino acid inserted at a specific position of a known signal peptide as a signal peptide. As a result, the present invention has been completed.

That is, the present invention includes the following inventions:
(A) A signal peptide for secreting a protein into the periplasm, wherein a basic amino acid is inserted into any of the N-terminal regions of the oligopeptide having the amino acid sequence set forth in SEQ ID NO: 2.

  (B) A signal for secreting a protein into the periplasm, wherein one residue of a basic amino acid is inserted between the second lysine and the third tyrosine of the oligopeptide having the amino acid sequence set forth in SEQ ID NO: 2. peptide.

  (C) The signal peptide according to (A) or (B), wherein the basic amino acid is lysine.

  (D) A plasmid for expressing the protein, comprising a polynucleotide encoding the signal peptide according to any one of (A) to (C) and a polynucleotide encoding the protein.

  (E) The plasmid according to (D), wherein the protein is a human Fc-binding protein.

  (F) A transformant obtained by transforming a host with the plasmid according to (D) or (E).

  (G) The transformant according to (F), wherein the host is Escherichia coli.

  (H) a step of expressing a protein from the transformant by culturing the transformant according to (F) or (G), and a step of isolating the protein from the transformant, A method for producing a protein.

  The present invention is described in detail below.

  The signal peptide of the present invention is a basic amino acid in any of the N-terminal regions of the natural PelB signal peptide (uniprot No. P0C1C1 region 1 to 22) consisting of the amino acid sequence shown in SEQ ID NO: 2. Is inserted. Here, the N-terminal region refers to the 4 amino acid residues on the N-terminal side (region from the first methionine to the fourth leucine) of the natural PelB signal peptide (SEQ ID NO: 2). Specifically, it is basic either between the first methionine and the second lysine, between the second lysine and the third tyrosine, between the third tyrosine and the fourth leucine. The signal peptide of the present invention can be obtained by inserting an amino acid. The number of basic amino acid insertions may be appropriately determined in the range of 1 to several amino acid residues in consideration of the properties of the protein secreted into the periplasm by the signal peptide of the present invention. As a specific example of the signal peptide of the present invention in which one basic amino acid residue is inserted between the second lysine and the third tyrosine of the natural PelB signal peptide (SEQ ID NO: 2), the natural PelB signal is described above. Oligopeptide (SEQ ID NO: 4) in which lysine is inserted between the second lysine and the third tyrosine of the peptide, or between the second lysine and the third tyrosine of the natural PelB signal peptide And oligopeptides in which histidine is inserted between the second lysine and the third tyrosine among the natural PelB signal peptides.

  The plasmid of the present invention comprises a polynucleotide (polynucleotide) in which a polynucleotide (polynucleotide B) encoding a signal peptide of the present invention is added to the 5 ′ end side of a polynucleotide (polynucleotide A) encoding a protein to be expressed in a host. C) is obtained by inserting into the appropriate position of the plasmid. In addition, when adding the polynucleotide B to the polynucleotide A, a polynucleotide encoding an arbitrary oligopeptide consisting of 1 to several amino acid residues is inserted between the polynucleotide A and the polynucleotide B as a linker. May be. The plasmid for inserting polynucleotide C is not particularly limited as long as it is stably present and replicable in the host to be transformed. When the host is E. coli, pET plasmid, pUC plasmid, pTrc plasmid, pCDF plasmid The pBBR plasmid can be exemplified. The appropriate position means a position where the replication function of the expression vector, a desired antibiotic marker, and a region related to transmissibility are not destroyed. In order to transform a host using the plasmid of the present invention, those skilled in the art may carry out the method usually used.

  There is no particular limitation on the protein produced using a transformant obtained by transforming a host using the plasmid of the present invention (hereinafter, simply referred to as the transformant of the present invention). Examples of such proteins include insulin and interferon. , Interleukins, antibodies, erythropoietin, human-derived proteins such as growth hormone, and their receptor proteins. The protein produced using the transformant of the present invention may be a complete protein, a protein composed only of a part important for the function of the protein, or the protein. One or more of the constituent amino acids may be deleted and / or inserted and / or substituted. Hereinafter, among the proteins that can be produced using the transformant of the present invention, the Fc-binding protein 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) (see FIG. 1), or the extracellular region of human FcγRIIIa (specifically, in the case of natural human FcγRIIIa, the 17th glycine to 208th glutamine in the amino acid sequence shown in SEQ ID NO: 22) (Refer to FIG. 4). However, it does not necessarily have to be the entire region of human FcγRI extracellular region or human FcγRIIIa extracellular region, and at least an antibody (immunoglobulin) among proteins (polypeptides) constituting human FcγRI extracellular region or human FcγRIIIa extracellular region The polypeptide of the area | region which can express the original function couple | bonded with Fc area | region of this should just be included. 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,
(Iii) a protein comprising an amino acid residue from at least the 17th glycine to the 192nd glutamine in the amino acid sequence of SEQ ID NO: 22,
(Iv) including at least the 17th glycine to the 192nd glutamine amino acid residue in the amino acid sequence shown in SEQ ID NO: 22, and at least one of the amino acid residues is substituted with another amino acid residue Inserted or deleted protein,
Can be given.

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: 21 ( Examples thereof include Fc-binding proteins (Japanese Patent Application Laid-Open No. 2014-027916) in which at least one substitution of 1) to (42) has occurred.
(1) 37th threonine of SEQ ID NO: 21 is replaced with isoleucine (2) 38th proline of SEQ ID NO: 21 is replaced with serine (3) 53rd leucine of SEQ ID NO: 21 is replaced with glutamine (4) SEQ ID NO: The 62nd glutamic acid of 21 is replaced with valine (5) The 63rd valine of SEQ ID NO: 21 is replaced with alanine or glutamic acid (6) The 66th leucine of SEQ ID NO: 21 is replaced with glutamine or proline (7) SEQ ID NO: 21 67 of serine is replaced with proline (8) 69th alanine of SEQ ID NO: 21 is replaced with valine or threonine (9) 71st serine of SEQ ID NO: 21 is replaced with threonine or leucine (10) 78th aspartic acid is replaced by glutamic acid (11) 81st isoleucine of SEQ ID NO: 21 is (12) The 84th serine of SEQ ID NO: 21 is replaced with threonine (13) The 88th phenylalanine of SEQ ID NO: 21 is replaced with tyrosine (14) The 95th glutamic acid of SEQ ID NO: 21 is replaced with aspartic acid ( 15) 119th histidine of SEQ ID NO: 21 replaced with glutamine (16) 127th valine of SEQ ID NO: 21 replaced with alanine (17) 146th arginine of SEQ ID NO: 21 replaced with lysine (18) SEQ ID NO: 21 The aspartic acid at position 147 was replaced with asparagine (19) The histidine at position 151 in SEQ ID NO: 21 was replaced with tyrosine (20) The threonine at position 178 in SEQ ID NO: 21 was replaced with alanine (21) The position 191 of SEQ ID NO: 21 Arginine is replaced with lysine (22) The 199th threonine of SEQ ID NO: 21 is (23) 200th leucine of SEQ ID NO: 21 replaced with methionine (24) 213th threonine of SEQ ID NO: 21 replaced with alanine (25) 216th valine of SEQ ID NO: 21 replaced with alanine (26 ) The 221st leucine of SEQ ID NO: 21 was replaced with arginine (27) The 229th serine of SEQ ID NO: 21 was replaced with asparagine (28) The 236th isoleucine of SEQ ID NO: 21 was replaced with lysine (29) 244th tyrosine is replaced with histidine (30) 253rd threonine of SEQ ID NO: 21 is replaced with alanine (31) 290th arginine of SEQ ID NO: 21 is replaced with glutamine (32) 293rd lysine of SEQ ID NO: 21 is replaced Substitution for asparagine (33) The 297th lysine of SEQ ID NO: 21 was substituted for glutamic acid. Substitution (34) The 306th proline of SEQ ID NO: 21 is replaced with threonine (35) The 34th glutamine of SEQ ID NO: 21 is substituted with arginine (36) The 45th glutamine of SEQ ID NO: 21 is substituted with lysine (37) The 82nd glutamine of No. 21 is replaced with proline (38) The 177th asparagine of SEQ ID NO: 21 is replaced with aspartic acid (39) The 213th threonine of SEQ ID NO: 21 is replaced with serine (40) 242 of SEQ ID NO: 21 The 41st glutamine is replaced with arginine (41) The 253rd threonine of SEQ ID NO: 21 is replaced with serine (42) The 271st glutamic acid of SEQ ID NO: 21 is replaced with aspartic acid, and as a specific example of the above (iv), Contains amino acid residues from position 17 to position 192 of the amino acid sequence of number 22. And an Fc-binding protein in which at least any one of the following (1) to (40) has occurred in the 17th to 192th amino acid residues (Japanese Patent Application No. 2013-202245) Can be given.
(1) 18th methionine of SEQ ID NO: 22 is replaced with arginine (2) 27th valine of SEQ ID NO: 22 is replaced with glutamic acid (3) 29th phenylalanine of SEQ ID NO: 22 is replaced with leucine or serine (4) 30th leucine of SEQ ID NO: 22 is replaced with glutamine (5) 35th tyrosine of SEQ ID NO: 22 is replaced with any of aspartic acid, glycine, lysine, leucine, asparagine, proline, serine, threonine, histidine (6) The 46th lysine of SEQ ID NO: 22 is replaced with isoleucine or threonine (7) The 48th glutamine of SEQ ID NO: 22 is replaced with histidine or leucine (8) The 50th alanine of SEQ ID NO: 22 is replaced with histidine (9) 51st tyrosine of number 22 is aspartic acid or histidine (10) The 54th glutamic acid of SEQ ID NO: 22 is replaced with aspartic acid or glycine (11) The 56th asparagine of SEQ ID NO: 22 is replaced with threonine (12) The 59th glutamine of SEQ ID NO: 22 is replaced with arginine (13) 61st phenylalanine of SEQ ID NO: 22 is replaced with tyrosine (14) 64th glutamic acid of SEQ ID NO: 22 is replaced with aspartic acid (15) 65th serine of SEQ ID NO: 22 is replaced with arginine (16) The 71st alanine of number 22 is replaced with aspartic acid (17) The 75th phenylalanine of SEQ ID NO: 22 is replaced with either leucine, serine or tyrosine (18) The 77th aspartic acid of SEQ ID NO: 22 is replaced with asparagine (19) The 78th alanine of SEQ ID NO: 22 becomes serine (20) The 82nd aspartic acid of SEQ ID NO: 22 is replaced with glutamic acid or valine (21) The 90th glutamine of SEQ ID NO: 22 is replaced with arginine (22) The 92nd asparagine of SEQ ID NO: 22 is replaced with serine ( 23) 93th leucine of SEQ ID NO: 22 is replaced with arginine or methionine (24) 95th threonine of SEQ ID NO: 22 is replaced with alanine or serine (25) 110th leucine of SEQ ID NO: 22 is replaced with glutamine (26 ) 115th Arginine of SEQ ID NO: 22 is replaced with glutamine (27) 116th Tryptophan of SEQ ID NO: 22 is replaced with leucine (28) 118th phenylalanine of SEQ ID NO: 22 is replaced with tyrosine (29) 119th lysine substituted with glutamic acid (30) sequence The 120th glutamic acid of No. 22 is substituted with valine (31) The 121st glutamic acid of SEQ ID No. 22 is substituted with aspartic acid or glycine (32) The 151st phenylalanine of SEQ ID No. 22 is substituted with serine or tyrosine (33) The 155th serine of No. 22 is replaced with threonine (34) The 163rd threonine of SEQ ID NO: 22 is replaced with serine (35) The 167th serine of SEQ ID NO: 22 is replaced with glycine (36) The 169th position of SEQ ID NO: 22 (37) 171th phenylalanine of SEQ ID NO: 22 is replaced with tyrosine (38) 180th asparagine of SEQ ID NO: 22 is replaced with lysine, serine or isoleucine (39) of SEQ ID NO: 22 185th threonine is replaced by serine (40) The 192st glutamine of 22 is replaced by lysine. The host used in preparing the transformant of the present invention includes animal cells typified by COS cells and CHO cells, Bacillus (Brevibacillus and Paenibacillus). And bacteria represented by Escherichia coli, yeasts represented by Saccharomyces, Pichia and Schizosaccharomyces, and filamentous fungi represented by Aspergillus. Preferably, Escherichia coli is used as a host. When the host is Escherichia coli and the protein is an Fc binding protein, the protein may be expressed by culturing the transformant of the present invention by the method disclosed in JP2012-034591A.

What is necessary is just to select suitably according to the form of expression, in order to collect | recover expressed proteins from the culture solution of the transformant of this invention. For example, when the expressed protein is expressed in the periplasm of the host cell, the host cell obtained by centrifuging the culture medium is suspended in an appropriate buffer, disrupted (physical disruption, disruption with drugs, etc.), and then centrifuged. By removing the crushing residue by separation, it is sufficient to obtain a cell-free extract containing the expressed protein. When the expressed protein leaks from the periplasm of the host cell to the culture supernatant, the culture solution is centrifuged. What is necessary is just to collect | recover the protein expressed from the culture supernatant obtained. When disrupting host cells 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, and 0.4% Triton X-100 (trade name) ), 0.5% CTAB (hexadecyltrimethylammonium bromide) and 50 mM CaCl ₂ , 50 mM Tris-HCl (pH 8.0) (Japanese Patent Laid-Open No. 2013-252099), BugBuster Protein extraction kit (manufactured by Takara Bio Inc.) It is good to crush using commercially available extraction reagents, such as.

  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 a carrier. As an example, TOYOPEARL CM-650, TOYOPEARL SP-650 (above, manufactured by Tosoh Corporation) CM Sepharose FastFlow (manufactured by GE Healthcare). 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 signal peptide of the present invention is an oligopeptide in which a basic amino acid is inserted into any of the N-terminal regions (regions from the first methionine to the fourth leucine) of the natural PelB signal peptides. By using this oligopeptide as a signal peptide for secreting proteins into the periplasm, it is possible to significantly improve the amount of recombinant protein produced compared to when using a natural PelB signal peptide, which is industrially useful. The production efficiency of a simple recombinant protein can be greatly improved.

The figure which shows the structure of natural type human FcγRI. The figure which compared the productivity of Fc binding protein when natural type PelB signal peptide was added to the N terminal side of Fc binding protein (human FcγRI origin) and when the signal peptide of the present invention was added. . The figure which compared the productivity of Fc binding protein when a natural type PelB signal peptide was added to the N terminal side of Fc binding protein (human FcγRIIIa), and when the signal peptide of the present invention was added. . The figure which shows the structure of natural type | mold human FcγRIIIa.

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

Example 1 Preparation of Expression Vector Expression is performed by inserting a polynucleotide encoding a natural PelB signal peptide (amino acid sequence: MKYLLPTAAAGLLLLAAQPAMA, SEQ ID NO: 2) into a plasmid pTrc99a (manufactured by GE Healthcare) by the following method. The vector pTrcpel was made.
(1) SEQ ID NO: 6 (5′-CATGAAATACCTGCTGCCGCACCGCTGCTGCTGTGTCTGCTGCCTCCGCCTGCC A double-stranded oligonucleotide PelBp1 was prepared by lowering the temperature every 1 ° C. over 1 minute until reaching 15 ° C. PelBp1 was kept at 15 ° C. until use.
(2) PelBp1 prepared in (1) was ligated to the plasmid pTrc99a previously digested with the restriction enzyme NcoI using DNA Ligation kit (manufactured by Takara Bio Inc.), and Escherichia coli JM109 strain (Takara Bio) was used using this ligation product. The product was transformed.
(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 pTrcpel was prepared using QIAprep Spin Miniprep kit (manufactured by Qiagen). Extracted.
(4) Big Dye Terminator Cycle Sequencing FS read Reaction kit (manufactured by Life Technologies) based on the chain terminator method for the polynucleotide encoding the PelB signal peptide and the surrounding region of the expression vector pTrcpel prepared in (3) 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, any one of the oligonucleotides described in SEQ ID NO: 8 (5′-TGTGGTATGGCTTGTGCAGG-3 ′) or SEQ ID NO: 9 (5′-TCGGCATGGGGTCAGGGTG-3 ′) was used as a sequencing primer.

  As a result of analysis, it was confirmed that it was as designed. The sequence of the polynucleotide encoding the natural PelB signal peptide synthesized in this example is shown in SEQ ID NO: 3.

Example 2 Preparation of Fc Binding Protein (FcRm68) Expression Vector (Part 1)
A polynucleotide encoding a polypeptide (FcRm68-CG) in which a cysteine tag (amino acid sequence: CG) is added to the C-terminal side of the Fc binding protein FcRm68 consisting of the amino acid sequence set forth in SEQ ID NO: 11 was prepared in Example 1. The protein expression vector was prepared by inserting the expression vector pTrcpel. FcRm68 is an Fc-binding protein FcRm60c (JP-A 2011-206046) consisting of a region from the 34th glutamine to the 307th aspartic acid in the amino acid sequence shown in SEQ ID NO: 10.
37th threonine of SEQ ID NO: 10 is isoleucine,
The 63rd valine of SEQ ID NO: 10 is glutamic acid,
The 69th alanine of SEQ ID NO: 10 is valine,
The 71st serine of SEQ ID NO: 10 is leucine,
84th serine of SEQ ID NO: 10 is threonine,
95th glutamic acid of SEQ ID NO: 10 is aspartic acid,
The 292nd proline of SEQ ID NO: 10 is lysine,
The 293rd glutamic acid of SEQ ID NO: 10 is lysine,
The 297th glutamine of SEQ ID NO: 10 is lysine,
301st histidine of SEQ ID NO: 10 to lysine
The 304th proline of SEQ ID NO: 10 is lysine,
Each is a substituted Fc binding protein. The amino acid sequence of the Fc binding protein FcRm60c 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, and 60 substitutions occurred in this region. (JP 2011-206046 A).
(1) A polynucleotide encoding the Fc-binding protein FcRm68 consisting of the amino acid sequence shown in SEQ ID NO: 11, and using the polynucleotide consisting of the sequence shown in SEQ ID NO: 12 as a template, SEQ ID NO: 13 PCR was carried out using oligonucleotides having the sequences described in 3 ′) and SEQ ID NO: 14 (5′-CCCGGAAGCTTAGCCGCCAGCTCCGGGGTTCTCTGTTGTTACCCAGTAC-3 ′) as PCR primers. 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 FcRm68-CG was obtained.

（２）得られたＦｃＲｍ６８−ＣＧをコードするポリヌクレオチドを制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化後、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した実施例１で作製した発現ベクターｐＴｒｃｐｅｌにライゲーションし、このライゲーション産物を用いて大腸菌Ｗ３１１０株を形質転換した。
（３）得られた形質転換体を、１００μｇ／ｍＬのカルベニシリンナトリウム（和光純薬社製）を含むＬＢ培地で培養後、ＱＩＡｐｒｅｐ Ｓｐｉｎ Ｍｉｎｉｐｒｅｐ ｋｉｔ（キアゲン社製）を用いて、ＦｃＲｍ６８−ＣＧをコードするポリヌクレオチドを含む発現ベクターｐＴｒｃＦｃＲｍ６８−ＣＧを抽出した。
（４）（３）で作製した発現ベクターｐＴｒｃＦｃＲｍ６８−ＣＧのうち、ＦｃＲｍ６８をコードするポリヌクレオチドおよびその周辺の領域について、実施例１（４）と同様の方法でヌクレオチド配列を解析した。

(2) The obtained polynucleotide encoding FcRm68-CG was digested with restriction enzymes NcoI and HindIII, and then ligated with the expression vector pTrcpel prepared in Example 1 previously digested with restriction enzymes NcoI and HindIII. Escherichia coli W3110 strain was used to transform it.
(3) The obtained transformant is cultured in an LB medium containing 100 μg / mL of carbenicillin sodium (manufactured by Wako Pure Chemical Industries, Ltd.), and then encoded using FIAR Spin Miniprep kit (manufactured by Qiagen) to encode FcRm68-CG. The expression vector pTrcFcRm68-CG containing the polynucleotide to be extracted was extracted.
(4) Among the expression vector pTrcFcRm68-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 1 (4).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcFcRm68-CG is shown in SEQ ID NO: 15, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 16, respectively. In SEQ ID NO: 15, the amino acid sequence of the PelB signal peptide is from the first methionine to the 22nd alanine (SEQ ID NO: 2), and the amino acid sequence of the Fc-binding protein FcRm68 is from the 25th glutamine to the 298th aspartic acid ( SEQ ID NO: 11) The amino acid sequence of the cysteine tag is from the 299th cysteine to the 300th glycine. In SEQ ID NO: 15, the amino acid sequence of FcRm68 (the 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 3 Preparation of Fc Binding Protein (FcRm68) Expression Vector (Part 2)
The signal peptide of the present invention in which lysine is inserted between the second lysine and the third tyrosine among the natural PelB signal peptides consisting of the amino acid sequence set forth in SEQ ID NO: 2 (amino acid sequence: MKKKYLLPTAAAGLLLLAAQPAMA, SEQ ID NO: 4) A polypeptide FcRm68-CG-V1 added to the N-terminal side of FcRm68-CG prepared in Example 2 was prepared by the method shown below.
(2) Using the expression vector pTrcFcRm68-CG prepared in Example 2 as a template, SEQ ID NO: 17 (5′-GGAAACAGACATCATAAAAAGTACCTGCTGCCGCACCGCTG-3 ′, and 28 nucleotides on the 3 ′ terminal side consist of the sequence described in SEQ ID NO: 4 (Corresponding to the region from the first to the 28th region of the polynucleotide sequence encoding the signal peptide of the present invention)) and an oligonucleotide having the sequence set forth in SEQ ID NO: 18 (5′-CACGCGTCGGCAGCAGGTACTTTTTCATGGTCTGTTTC-3 ′) as a primer, The following reaction was performed. After preparing a reaction solution having the composition shown in Table 1, the reaction solution was defined as one cycle of 98 ° C. for 10 seconds for the first step, 55 ° C. for 5 seconds for the second step, and 72 ° C. for 5 minutes for 5 minutes. This reaction was performed by repeating 16 cycles. The resulting product was digested with the restriction enzyme DpnI to decompose the template plasmid, and a plasmid containing a polynucleotide encoding FcRm68-CG-V1 was obtained.
(2) Escherichia coli W3110 was transformed with the obtained plasmid containing a polynucleotide encoding FcRm68-CG-V1.
(3) The obtained transformant is cultured in an LB medium containing 100 μg / mL of carbenicillin sodium (manufactured by Wako Pure Chemical Industries, Ltd.), and using QIAprep Spin Miniprep kit (manufactured by Qiagen), FcRm68-CG-V1 The expression vector pTrcFcRm68-CG-V1 containing the encoding polynucleotide was extracted.
(4) The desired sequence was confirmed by the sequence analysis described in Example 1 (4).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcFcRm68-CG-V1 is shown in SEQ ID NO: 19, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 20, respectively. In SEQ ID NO: 19, the amino acid sequence of the signal peptide of the present invention (SEQ ID NO: 4) is from the first methionine to the 23rd alanine, and the amino acid sequence of the Fc-binding protein FcRm68 is from the 26th glutamine to the 299th aspartic acid. The sequence (SEQ ID NO: 11), from the 300th cysteine to the 301st glycine is a cysteine tag.

Example 4 Expression of Fc-binding protein (1)
(1) E. coli W3110 strain was transformed with the expression vector pTrcFcRm68-CG-V1 prepared in Example 3, and 2YT containing 100 μg / mL carbenicillin sodium (Tryptone: 16 g / L, yeast extract: 10 g / L, Sodium chloride: 5 g / L) Inoculated in a liquid medium and precultured by aerobic shaking culture at 37 ° C. overnight.
(2) 600 μL of the preculture was inoculated into 20 mL of 2YT liquid medium containing 100 μg / mL carbenicillin sodium, and cultured with shaking at 37 ° C. aerobically.
(3) Three hours after the start of the culture, the culture temperature was changed to 20 ° C., and after 30 minutes of shaking culture, IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 0.05 mM. The culture was aerobically shaken overnight at 20 ° C.
(4) After completion of the culture, the cells were collected by centrifugation, and a protein extract was prepared using BugBuster Protein extraction kit (Takara Bio Inc.).
(5) The concentration of the Fc binding protein in the protein extract prepared in (4) was measured for the antibody binding activity using the ELISA method shown below, and compared with the value in the Fc binding protein at a known concentration. It was measured.
(5-1) A gamma globulin preparation which is a human antibody (manufactured by Chemical and Serum Therapy Research Laboratories) was immobilized in 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 Blocking was performed with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5% (w / v) BSA (Sigma Aldrich) and 150 mM NaCl.
(5-2) Fc-binding protein prepared after washing with washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% (w / v) Tween 20 and 150 mM NaCl) The solution containing was reacted with immobilized gamma globulin (1 hour at 30 ° C.).
(5-3) After completion of the reaction, 100 μL / well of Anti-FcγRI antibody (manufactured by R & D Systems) diluted with 50 ng / mL was washed with the washing buffer.
(5-4) After reacting at 30 ° C. for 1 hour, Horse radish Peroxidase (HRP) -labeled Anti-mouse-IgG antibody (manufactured by BETHYL) diluted with 50 ng / mL was washed with 100 μL / well. Added.
(5-5) After reacting at 30 ° C. for 1 hour, the plate is washed with the washing buffer, TMB Peroxidase Substrate (manufactured by KPL) is added at 50 μL / well, and 1M phosphoric acid aqueous solution is added at 50 μL / well for color reaction. Stop and measure the absorbance at 450 nm.

  The concentration of Fc-binding protein after culture was measured from a calibration curve using purified Fc-binding protein of known concentration by ELISA. The productivity of Fc-binding protein per cell was calculated by dividing the concentration of Fc-binding protein after culture by the absorbance at 600 nm measured as the amount of cells at the end of the culture. The results are shown in FIG.

Comparative Example 1 Expression of Fc binding protein (2)
Using the Fc-binding protein expression vector pTrcFcRm68-CG containing the natural PelB signal peptide (SEQ ID NO: 2) prepared in Example 2, E. coli strain W3110 was transformed and cultured in the same manner as in Example 4. The productivity of the Fc binding protein was calculated. The results are shown in FIG.

Example 5 Preparation of Fc Binding Protein (FcR5a) Expression Vector In the expression vector pTrcFcRm68-CG-V1 prepared in Example 3, the region of the polynucleotide encoding the Fc binding protein is the amino acid sequence set forth in SEQ ID NO: 23. An expression vector pTrcFcR5a-V1 substituted with a polynucleotide encoding Fc-binding protein FcR5a comprising: FcR5a is an Fc-binding protein consisting of a region from the 17th glycine to the 192nd glutamine in the amino acid sequence shown in SEQ ID NO: 22,
27th valine of SEQ ID NO: 22 to glutamic acid,
Asparagine of the 35th tyrosine of SEQ ID NO: 22,
The 75th phenylalanine of SEQ ID NO: 22 is used as leucine and the 92nd asparagine of SEQ ID NO: 22 is used as serine,
Glycine is the 121st glutamic acid of SEQ ID NO: 22,
Each is a substituted Fc-binding protein (Japanese Patent Application No. 2013-202245).
(1) An expression vector pETmalE (Japanese Patent Application Laid-Open No. 2011-206046), which is a polynucleotide encoding the Fc-binding protein FcR5a consisting of the amino acid sequence shown in SEQ ID NO: 23, which is the sequence shown in SEQ ID NO: 24 The expression vector pET-FcR5a (Japanese Patent Application No. 2013-202245) was obtained.
(2) The obtained expression vector pET-FcR5a was digested with restriction enzymes NcoI and HindIII, and the digested product was obtained by removing the vector portion.
(3) The pTrcFcRm68-CG-V1 obtained in Example 3 was digested with restriction enzymes NcoI and HindIII in advance and the vector portion was purified, and the digested product prepared in (2) was ligated, Escherichia coli W3110 strain was used to transform it.
(4) The obtained transformant is cultured in an LB medium containing 100 μg / mL of carbenicillin sodium (manufactured by Wako Pure Chemical Industries, Ltd.), and is described in SEQ ID NO: 4 using QIAprep Spin Miniprep kit (manufactured by Qiagen). An expression vector pTrcFcR5a-V1 containing a polynucleotide encoding the polypeptide FcR5a-V1 obtained by adding the signal peptide of the present invention consisting of a sequence to the N-terminal side of the Fc binding protein FcR5a was extracted.

  The sequence analysis described in Example 1 (4) was performed on the expression vector pTrcFcR5a-V1, and as a result, it was confirmed that the desired sequence was obtained.

Example 6 Expression of Fc-binding protein (Part 3)
Escherichia coli W3110 was transformed with the expression vector pTrcFcR5a-V1 prepared in Example 5, and the obtained transformant was cultured in the same manner as in Examples 4 (1) to (3). A protein extract was prepared in the same manner as in 4 (4), and the concentration of the Fc binding protein was measured in the same manner as in Example 4 (5) to calculate the productivity of the Fc binding protein ( However, the Anti-FcγRI antibody in Example 4 (5-3) was an Anti-FcγRIII antibody). The results are shown in FIG.

Comparative Example 2 Expression of Fc binding protein (4)
Of the Fc-binding protein expression vector pTrcFcRm68-CG containing the natural PelB signal peptide prepared in Example 2, the region of the polynucleotide encoding the Fc-binding protein is the Fc binding consisting of the amino acid sequence set forth in SEQ ID NO: 23 An expression vector pTrcFcR5a substituted with a polynucleotide encoding the sex protein FcR5a (SEQ ID NO: 24) was prepared in the same manner as in Example 5, and the productivity of Fc-binding protein was calculated in the same manner as in Example 6. The results are shown in FIG.

  2 and 3, the signal peptide of the present invention in which a basic amino acid is inserted between the second and third of the natural PelB signal peptide is used as a signal peptide for secreting the recombinant protein into the periplasm (Examples 4 and 3). 6) Thus, it can be seen that the productivity of the protein is improved as compared with the case of using the natural PelB signal peptide (Comparative Examples 1 and 2).

Claims (8)

A signal peptide for secreting a protein into the periplasm, wherein a basic amino acid is inserted into any of the N-terminal regions of the oligopeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2. A signal peptide for secreting a protein into the periplasm, wherein one residue of a basic amino acid is inserted between the second lysine and the third tyrosine of the oligopeptide having the amino acid sequence set forth in SEQ ID NO: 2. The signal peptide according to claim 1 or 2, wherein the basic amino acid is lysine. The plasmid for expressing the said protein containing the polynucleotide which codes the signal peptide in any one of Claim 1 to 3, and the polynucleotide which codes protein. The plasmid according to claim 4, wherein the protein is a human Fc-binding protein. A transformant obtained by transforming a host with the plasmid according to claim 4 or 5. The transformant according to claim 6, wherein the host is Escherichia coli. A method for producing the protein, comprising a step of expressing the protein from the transformant by culturing the transformant according to claim 6 or 7, and a step of isolating the protein from the transformant.

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