JP6136452B2 - Oligopeptide for immobilizing protein on carrier - Google Patents

Description

  The present invention relates to an oligopeptide for immobilizing a protein on an insoluble carrier with high efficiency.

  Chromatography is widely used to separate target substances from a mixture, such as ion exchange chromatography that separates based on the difference in charge, hydrophobic interaction chromatography that separates based on the difference in hydrophobicity, And gel filtration chromatography that separates based on molecular size. Among chromatography, affinity chromatography is an adsorption chromatography obtained by immobilizing a substance that can specifically bind to a target substance (hereinafter referred to as a ligand) on an insoluble carrier, and has high purity in one step. Purification is possible.

  Affinity chromatography is particularly used for purification of pharmaceuticals (antibody pharmaceuticals) mainly composed of antibodies (immunoglobulins), and affinity chromatography using an adsorbent with protein A as a ligand is frequently used. On the other hand, Fc receptors, which are a group of protein molecules that bind to the Fc region of an antibody, and Fc receptor partial regions (hereinafter collectively referred to as Fc binding proteins) having the ability to bind to Fc regions are also referred to as protein A. Similarly, an antibody can be used as a ligand capable of specifically adsorbing (Patent Document 1).

  When a protein is immobilized on a carrier as a ligand, an active functional group (for example, epoxy) in which a highly reactive amino acid side chain (for example, an ε-amino group possessed by a lysine residue) present on the surface of the ligand is introduced into the carrier. Generally, a method of immobilizing on a carrier by covalent bonding with a group or N-hydroxysuccinimide (NHS group) is generally used. However, when there are a plurality of lysine residues in the ligand, the ligand is immobilized on the carrier at random. In other words, there is a possibility that a part or all of the binding region of the ligand with the target substance may be immobilized on the carrier, and as a result, the immobilized ligand cannot bind to the target substance. Was.

JP 2010-126436 A JP 2011-206046 A

  In order to solve the above-mentioned problems, cysteine, which is an amino acid having a thiol group that is more reactive than the ε-amino group, is introduced at the N-terminal side or C-terminal side of the protein to increase the selectivity of the reaction (orientation) Control). However, when a protein is immobilized on an insoluble carrier using a thiol group, the protein immobilization rate is not always sufficient.

  An object of the present invention is to provide an oligopeptide that can be immobilized on an insoluble carrier with high efficiency while controlling the orientation of the protein.

  As a result of intensive studies to solve the above problems, the present inventors have found that the amino acid residue on the N-terminal side is cysteine, and the two amino acid residues on the C-terminal side are cysteine-glycine. It has been found that by adding an oligopeptide to the protein, the protein can be immobilized on an insoluble carrier with high efficiency while controlling the orientation of the protein, and the present invention has been completed.

That is, the first aspect of the present invention is:
An oligopeptide consisting of 5 to 8 amino acid residues for immobilizing a protein on an insoluble carrier,
At least the N-terminal amino acid residue is cysteine, and the C-terminal two amino acid residues are cysteine-glycine.

  The second aspect of the present invention is the oligopeptide according to the first aspect, comprising the amino acid sequence of any one of SEQ ID NOs: 14, 18, 22, 26, 30, 34, 38, 42, and 46. It is.

  The third aspect of the present invention is an oligopeptide-added protein in which the oligopeptide according to the first or second aspect is added to the N-terminal side or C-terminal side of the protein.

  The fourth aspect of the present invention is the oligopeptide-added protein according to the third aspect, wherein the protein is an Fc-binding protein.

  The fifth aspect of the present invention is the amino acid sequence according to the fourth aspect, comprising the amino acid sequence of any one of SEQ ID NOs: 16, 20, 24, 28, 32, 36, 40, 44, 48, 54. It is an oligopeptide-added protein.

  A sixth aspect of the present invention is a protein adsorbent obtained by immobilizing the oligopeptide-added protein according to any of the third to fifth aspects on an insoluble carrier.

  A seventh aspect of the present invention is a protein purification method using the adsorbent according to the sixth aspect.

  The eighth aspect of the present invention is a polynucleotide encoding the oligopeptide-added protein according to any one of the third to fifth aspects.

  The ninth aspect of the present invention is an expression vector comprising the polynucleotide according to the eighth aspect.

  The tenth aspect of the present invention is a transformant obtained by transforming a host with the expression vector according to the ninth aspect.

  An eleventh aspect of the present invention is a method for producing an oligopeptide-added protein, wherein the transformant according to the tenth aspect is cultured, and the oligopeptide-added protein is recovered from the obtained culture solution.

  Hereinafter, the present invention will be described in detail.

  In the present invention, as an oligopeptide for immobilizing a protein on an insoluble carrier, the amino acid residue on the N-terminal side is cysteine (C), and the two amino acid residues on the C-terminal side are cysteine-glycine (CG). An oligopeptide consisting of 5 to 8 amino acid residues is used. As an example of the oligopeptide of the present invention, CTRCG (SEQ ID NO: 14), CKTCG (SEQ ID NO: 18), CRNDTCG (SEQ ID NO: 22), CRSDPCG (SEQ ID NO: 26), CLGESVCG (SEQ ID NO: 30), CIEGEPCG (SEQ ID NO: 34) CHNGKCG (SEQ ID NO: 38), CGSGKCG (SEQ ID NO: 42), and CDGGKCG (SEQ ID NO: 46).

  The protein to which the oligopeptide of the present invention is added is not particularly limited as long as it can be used as a ligand for affinity chromatography. Examples thereof include immunoglobulins such as protein A, protein G, protein L, and Fc-binding protein ( Antibodies) Proteins that can be used as ligands for purification. Hereinafter, among the proteins, Fc binding proteins will be described in detail.

In this specification, human Fc-binding protein constitutes the extracellular region of human FcγRI (specifically, in the case of natural human FcγRI, the region from the 16th to the 292nd of the amino acid sequence described in SEQ ID NO: 7). It refers to the protein. 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 of SEQ ID NO: 7,
(Ii) including at least the 16th glutamine to 289th valine amino acid residues in the amino acid sequence of SEQ ID NO: 7, and replacing one or more of the amino acid residues with another amino acid residue , Inserted or deleted proteins.

Specific examples of (ii) include the human Fc-binding protein disclosed in JP2011-206046 (Patent Document 2) and the amino acids 34 to 307 of the amino acid sequence set forth in SEQ ID NO: 56. An Fc-binding protein comprising a residue and having at least one of the following substitutions (1) to (42) occurring in the 34th to 307th amino acid residues (Japanese Patent Application No. 2012-270375) Issue).
(1) 37th threonine of SEQ ID NO: 56 is replaced with isoleucine (2) 38th proline of SEQ ID NO: 56 is replaced with serine (3) 53rd leucine of SEQ ID NO: 56 is replaced with glutamine (4) SEQ ID NO: The 62nd glutamic acid of 56 is replaced with valine (5) The 63rd valine of SEQ ID NO: 56 is replaced with alanine or glutamic acid (6) The 66th leucine of SEQ ID NO: 56 is replaced with glutamine or proline (7) SEQ ID NO: 56 67 of serine is replaced with proline (8) 69th alanine of SEQ ID NO: 56 is replaced with valine or threonine (9) 71st serine of SEQ ID NO: 56 is replaced with threonine or leucine (10) of SEQ ID NO: 56 78th aspartic acid was replaced by glutamic acid (11) 81st isoleucine of SEQ ID NO: 56 was (12) The 84th serine of SEQ ID NO: 56 is replaced with threonine (13) The 88th phenylalanine of SEQ ID NO: 56 is replaced with tyrosine (14) The 95th glutamic acid of SEQ ID NO: 56 is replaced with aspartic acid ( 15) 119th histidine of SEQ ID NO: 56 replaced with glutamine (16) 127th valine of SEQ ID NO: 56 replaced with alanine (17) 146th arginine of SEQ ID NO: 56 replaced with lysine (18) SEQ ID NO: 56 The aspartic acid at position 147 was replaced with asparagine (19) The histidine at position 151 in SEQ ID NO: 56 was replaced with tyrosine (20) The threonine at position 178 in SEQ ID NO: 56 was replaced with alanine (21) The position 191 of SEQ ID NO: 56 Arginine is replaced with lysine (22) The 199th threonine of SEQ ID NO: 56 is (23) 200th leucine of SEQ ID NO: 56 replaced with methionine (24) 213th threonine of SEQ ID NO: 56 replaced with alanine (25) 216th valine of SEQ ID NO: 56 replaced with alanine (26 ) The 221st leucine of SEQ ID NO: 56 was replaced with arginine (27) The 229th serine of SEQ ID NO: 56 was replaced with asparagine (28) The 236th isoleucine of SEQ ID NO: 56 was replaced with lysine (29) 244th tyrosine is replaced with histidine (30) 253rd threonine of SEQ ID NO: 56 is replaced with alanine (31) 290th arginine of SEQ ID NO: 56 is replaced with glutamine (32) 293rd lysine of SEQ ID NO: 56 is Substitution for asparagine (33) The 297th lysine of SEQ ID NO: 56 was substituted for glutamic acid. Substitution (34) The 306th proline of SEQ ID NO: 56 is replaced with threonine (35) The 34th glutamine of SEQ ID NO: 56 is substituted with arginine (36) The 45th glutamine of SEQ ID NO: 56 is substituted with lysine (37) The 82nd glutamine of No. 56 is replaced with proline (38) The 177th asparagine of SEQ ID NO: 56 is substituted with aspartic acid (39) The 213th threonine of SEQ ID NO: 56 is substituted with serine (40) 242 of SEQ ID NO: 56 The 253rd threonine of SEQ ID NO: 56 is replaced with serine (42) the 271st glutamic acid of SEQ ID NO: 56 is replaced with aspartic acid; the oligo of the present invention on the N-terminal side or C-terminal side; A polynucleotide encoding a protein to which a peptide is added (hereinafter simply referred to as the polynucleotide of the present invention). As a production method of renucleotide,
(I) a method of artificially synthesizing a polynucleotide containing the nucleotide sequence by converting the amino acid sequence of the protein to which the oligopeptide of the present invention has been added to the N-terminal side or C-terminal side into a nucleotide sequence,
(II) a method of adding an oligonucleotide encoding the oligopeptide of the present invention to a polynucleotide encoding a protein by a genetic engineering technique using a DNA amplification method such as a PCR method,
Can be illustrated. In addition, when converting an amino acid sequence into a nucleotide sequence, it is preferable to convert in consideration of the codon usage frequency in the host to be transformed. For example, when the host is Escherichia coli, AGA / AGG / CGG / CGA is used for arginine (Arg), ATA is used for isoleucine (Ile), CTA is used for leucine (Leu), and GGA is used for glycine (Gly). In proline (Pro), since CCC is used less frequently (because it is a so-called rare codon), it may be converted to avoid those codons. For analysis of codon usage frequency, a public database (for example, Codon Usage Database on the homepage of Kazusa DNA Research Institute) can be used. Further, when producing the polynucleotide of the present invention by the method of (I) or (II), a polynucleotide encoding a signal peptide may be added to the 5 ′ end of the polynucleotide encoding the protein, When Escherichia coli is E. coli, examples of the signal peptide include signal peptides that secrete proteins into the periplasm such as PelB (SEQ ID NO: 1), DsbA, MalE, and TorT.

  When transforming a host using the polynucleotide of the present invention, the polynucleotide of the present invention itself may be used. However, in terms of improving transformation efficiency, expression vectors (for example, prokaryotic cells and eukaryotic cells) It is preferable to use those in which the polynucleotide of the present invention is inserted at an appropriate position in bacteriophage, cosmid, plasmid, etc., which are usually used for conversion. The expression vector is not particularly limited as long as it is stable and can be replicated in the host to be transformed. When Escherichia coli is used as a host, pET plasmid vector, pUC plasmid vector, pTrc plasmid vector, pCDF plasmid Examples of the vector include a pBBR plasmid vector. 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. When inserting the polynucleotide of the present invention into the expression vector, it is preferable to insert it in a state linked to a functional polynucleotide such as a promoter required for expression. Examples of the promoter include trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, λ phage λPL promoter, λPR promoter when the host is Escherichia coli. . In order to transform a host with an expression vector (including the polynucleotide of the present invention) into which the polynucleotide of the present invention prepared by the above method is inserted, a person skilled in the art may carry out the method usually used.

  When producing a protein having the oligopeptide of the present invention added to the N-terminal side or C-terminal side (hereinafter referred to as the oligopeptide-added protein of the present invention), it was transformed with an expression vector containing the polynucleotide of the present invention. It can be produced by culturing a host (transformant). Specifically, the transformant may be cultured, and the oligopeptide-added protein of the present invention may be recovered from the culture. In this specification, the culture includes not only the cultured transformant cells themselves but also the medium used for the culture. In order to produce a protein using the transformant of the present invention, it may be cultured in a medium suitable for culturing the host of the transformant. When the host is E. coli, LB (supplemented with necessary nutrients) Luria-Bertani) medium is an example of a preferable medium. In order to selectively grow transformants depending on the presence or absence of introduction of a vector containing the polynucleotide of the present invention, it is preferable to add a drug corresponding to the drug resistance gene contained in the vector to the medium and culture. For example, when the vector contains a kanamycin resistance gene, kanamycin may be added to the medium. In addition to the carbon, nitrogen and inorganic salt sources, an appropriate nutrient source may be added to the medium, and if desired, the medium is selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate and dithiothreitol. One or more reducing agents may be included. Furthermore, a reagent that promotes protein secretion from the transformant to the culture solution, such as glycine, may be added. Specifically, when the host is Escherichia coli, glycine is 2% (w / v) or less with respect to the medium. Is preferably added. When the host is Escherichia coli, the culture temperature is generally 10 ° C. to 40 ° C., preferably 20 ° C. to 37 ° C., more preferably around 25 ° C., but may be selected depending on the characteristics of the protein to be expressed. When the host is Escherichia coli, the pH of the medium is from pH 6.8 to pH 7.4, preferably around pH 7.0. When an inducible promoter is included in the expression vector, it is preferable to induce under conditions that allow the oligopeptide-added protein of the present invention to be expressed well. An example of the inducer is IPTG (isopropyl-β-D-thiogalactopyranoside). When the host is Escherichia coli, the turbidity (absorbance at 600 nm) of the culture solution is measured, and when it becomes about 0.5 to 1.0, an appropriate amount of IPTG is added, and then the culture is continued. Expression of oligopeptide-added protein can be induced. The addition concentration of IPTG may be appropriately selected from the range of 0.005 to 1.0 mM, but is preferably in the range of 0.01 to 0.5 mM. Various conditions relating to the IPTG induction may be performed under conditions well known in the art.

  In order to recover the oligopeptide-added protein of the present invention from the culture medium of the transformant of the present invention, a recovery method may be appropriately selected depending on the expression form of the protein. When expressed in the culture supernatant of the culture solution, the cells are separated by centrifugation or the like, and the oligopeptide-added protein of the present invention may be recovered from the obtained culture supernatant. On the other hand, when expressed in cells (including periplasm in prokaryotes), after collecting the cells by centrifugation, the cells are disrupted by adding an enzyme treatment agent or a surfactant, What is necessary is just to collect | recover the oligopeptide addition protein of this invention.

  In order to separate and purify the protein from the extracted solution containing the oligopeptide-added protein of the present invention, a method known in the art may be used, and an example is separation and purification using liquid chromatography. Liquid chromatography includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, and the like, and the oligopeptide addition of the present invention can be performed by performing a purification operation by combining these chromatography. Proteins can be prepared with high purity.

  The protein adsorbent of the present invention (hereinafter simply referred to as the adsorbent of the present invention) can be prepared by immobilizing the oligopeptide-added protein of the present invention on an insoluble carrier. The insoluble carrier used for producing the adsorbent of the present invention is not particularly limited, but is a polysaccharide such as agarose, alginate (alginate), carrageenan, chitin, cellulose, dextrin, dextran, starch, or polyvinyl alcohol, polymethacrylate, Synthetic polymer gels such as poly (2-hydroxyethyl methacrylate) and polyurethane are preferred. Examples of the preferred carrier include polymethacrylate gels introduced with hydroxyl groups such as Toyopearl (manufactured by Tosoh Corporation), agarose gels such as Sepharose (manufactured by GE Healthcare), and cellulose gels such as Cellufine (manufactured by JNC). . There is no particular limitation on the shape of the carrier, and it may be granular or non-particulate, porous or non-porous.

  When immobilizing the oligopeptide-added protein of the present invention on an insoluble carrier, an active group such as an N-hydroxysuccinimide (NHS) activated ester group, an epoxy group, a maleimide group, a tresyl group, or a formyl group haloacetamide is used. And may be immobilized by covalent bonding to the carrier. As the carrier having an active group, a commercially available carrier may be used as it is, or it may be prepared by introducing an active group on the surface of the carrier under appropriate reaction conditions. Commercially available carriers having an active group include TOYOPEARL AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M (both manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4F, Examples thereof include 6B (both manufactured by GE Healthcare) and SulfoLink Coupling Resin (manufactured by Thermo Scientific).

  Examples of the method for introducing an active group on the surface of the carrier include a method in which one of compounds having two or more active sites reacts with a hydroxyl group, an epoxy group, a carboxyl group, an amino group or the like present on the surface of the carrier.

  Examples of the compound that introduces an epoxy group into the hydroxyl group or amino group on the surface of the carrier include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.

  Examples of the compound that introduces an epoxy group on the surface of the carrier and introduces a carboxyl group on the surface of the carrier as described above include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3 -Aminopropionic acid, 4-aminobutyric acid, 6-aminohexanoic acid can be illustrated.

  As a compound for introducing a maleimide group into a hydroxyl group, epoxy group, carboxyl group or amino group present on the surface of the carrier, N- (ε-maleimidocaproic acid) hydrazide, N- (ε-maleimidopropionic acid) hydrazide, 4- [4 -N-maleimidophenyl] acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1- (4-aminophenyl) maleimide, 1- (3-aminophenyl) maleimide, 4 -(Maleimido) phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, (N- [α-maleimidoacetoxy] succinimide ester), (m-maleimidobenzoyl) N -Hydroxysuccinimide ester, ( Cuccinimidyl-4- [maleimidomethyl] cyclohexane-1-carbonyl- [6-aminohexanoic acid]), (succinimidyl-4- [maleimidomethyl] cyclohexane-1-carboxylic acid), (p-maleimidobenzoyl) N-hydroxysuccinimide Examples of the ester include (m-maleimidobenzoyl) N-hydroxysuccinimide ester.

  Compounds that introduce a haloacetyl group into the hydroxyl group or amino group present on the surface of the carrier include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, Iodoacetic anhydride, 2- (iodoacetamido) acetic acid-N-hydroxysuccinimide ester, 3- (bromoacetamido) propionic acid-N-hydroxysuccinimide ester, 4- (iodoacetyl) aminobenzoic acid-N-hydroxysuccinimide ester It can be illustrated.

  Alternatively, an active group may be introduced by reacting a ω-alkenyl alkanglycidyl ether with a hydroxyl group or amino group present on the surface of the carrier and then halogenating the ω-alkenyl moiety with a halogenating agent. Examples of ω-alkenyl alkanglycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether. Examples of halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide. it can.

  As another example of the method for introducing an active group on the surface of the carrier, there is a method for introducing an activating group into the carboxyl group on the surface of the carrier using a condensing agent and an additive. Examples of the condensing agent include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of the additive include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenztriazole.

  The amount of active group introduced into the carrier is preferably 1 nmol to 1 mmol, more preferably 1 μmol to 100 μmol per mL of carrier.

  When immobilizing the oligopeptide-added protein of the present invention on an insoluble carrier, the protein may be dissolved in a buffer solution and reacted with an active group of the carrier. As the buffer solution, an acetate buffer solution, a phosphate buffer solution, Examples thereof include MES buffer, HEPES buffer, Tris buffer, and borate buffer. The reaction temperature for immobilization may be appropriately set in the range of 5 ° C. to 50 ° C. in consideration of the reactivity of the active group and the stability of the protein of the present invention, preferably 10 ° C. to 35 ° C. It is a range.

  The protein purification method using the adsorbent of the present invention is not particularly limited, but the purification method by chromatography using the column obtained by filling the empty column with the adsorbent of the present invention purifies the target substance with high purity. It is preferable in that it can be performed. Examples of the solution containing the target substance to be purified by the adsorbent of the present invention include a culture solution of a host that produces the target substance, and a culture supernatant from which the host cells have been removed by centrifugation or the like. The size of the column (packing amount of the adsorbent of the present invention) may be appropriately determined in consideration of the processing amount of the purified raw material.

  When the solution containing the target substance is added to the column packed with the adsorbent of the present invention, the solution may be added using a liquid feeding means such as a pump in consideration of the liquid feeding amount. In addition, the column packed with the adsorbent of the present invention is preferably equilibrated in advance using an appropriate buffer before the antibody is added, because the antibody can be purified with higher purity. Examples of the buffer solution include a buffer solution containing an inorganic salt as a component, such as a phosphate buffer solution. The pH of the buffer solution is pH 3 to 10, preferably pH 5 to 8. In order to elute the target substance adsorbed on the adsorbent, it is only necessary to weaken the interaction between the protein immobilized on the adsorbent and the target substance. Examples thereof include temperature change or salt concentration change. When the target substance is an antibody, a change in pH due to a buffer can be exemplified, and examples of the buffer used include a citrate buffer, a glycine hydrochloride buffer, and an acetate buffer. The pH of the buffer solution may be set in a range that does not impair the function of the antibody, and is preferably pH 3 to 6, more preferably pH 3.3 to 4.0.

  The present invention provides an oligopeptide for immobilizing a protein on an insoluble carrier, in which 5 to 8 amino acid residues are obtained, in which the amino acid residue on the N-terminal side is cysteine and the 2 amino acid residues on the C-terminal side are cysteine-glycine. It is characterized by using an oligopeptide comprising a group. According to the present invention, the protein can be immobilized on a carrier with high efficiency while controlling the orientation of the protein. Furthermore, by adding the oligopeptide of the present invention to the N-terminal side or C-terminal side of the protein, there is also a new effect that the production amount of the protein is improved.

The figure which shows the result of having evaluated the difference in the immobilization rate to an insoluble support | carrier by the difference in the oligopeptide added to Fc binding protein. The figure which shows the performance evaluation result of each Fc binding protein fixed gel.

  Hereinafter, the present invention will be described in more detail using examples in which the oligopeptide of the present invention is added to an Fc-binding protein, but the present invention is not limited to these examples.

Example 1 Preparation of Expression Vector An expression vector was inserted by inserting a polynucleotide encoding a PelB signal peptide (amino acid sequence: MKYLLPTAAAGLLLLAAQPAMA, SEQ ID NO: 1) into an expression vector pTrc99a (manufactured by GE Healthcare) by the following method. pTrcpel was prepared.
(1) SEQ ID NO: 2 (5'-CATGAAATACCTGCTGCCGCACCGCTGCTGCTGCGTCTGCTGCCTCAGCCGGCGGATGGC-3 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 expression vector pTrc99a previously digested with the restriction enzyme NcoI using a DNA ligation kit (manufactured by Takara Bio Inc.), and E. coli JM109 strain (Takara Bio Inc.) was used. Made).
(3) After culturing the obtained transformant in an LB medium containing 100 μg / mL of carbenicillin sodium (manufactured by Wako Pure Chemical Industries, Ltd.), the expression vector pTrcpel is extracted using QIAprep Spin Miniprep kit (manufactured by Qiagen). did.
(4) Big Dye Terminator Cycle Sequencing FS read Reaction kit (manufactured by Life Science) 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 using a fully automatic DNA sequencer ABI Prism 3700 DNA analyzer (manufactured by Life Sciences). In the analysis, any one of the oligonucleotides described in SEQ ID NO: 4 (5′-TGTGGTATGGCTTGTGCAGG-3 ′) or SEQ ID NO: 5 (5′-TCGGCATGGGGTCAGGGTG-3 ′) was used as a sequencing primer.

  As a result of analysis of the nucleotide sequence, it was confirmed as designed.

Comparative Example 1 Preparation of Fc-binding protein expression vector to which cysteine tag was added (part 1)
A polynucleotide encoding a polypeptide (FcRm68-CG) having a cysteine tag (amino acid sequence: CG) added to the C-terminal side of the Fc binding protein FcRm68 consisting of the amino acid sequence shown in SEQ ID NO: 8 was prepared in Example 1. An FcRm68-CG expression vector was prepared by inserting the expression vector pTrcpel. FcRm68 is an Fc-binding protein FcRm60c consisting of a region from the 34th glutamine to the 307th aspartic acid in the amino acid sequence shown in SEQ ID NO: 6 (JP 2011-206046 A, Patent Document 2).
37th threonine of SEQ ID NO: 6 is isoleucine,
63rd valine of SEQ ID NO: 6 is glutamic acid,
69th alanine of SEQ ID NO: 6 is valine,
The 71st serine of SEQ ID NO: 6 is leucine,
84th serine of SEQ ID NO: 6 is threonine,
95th glutamic acid of SEQ ID NO: 6 is aspartic acid,
The 292nd proline of SEQ ID NO: 6 is lysine,
The 293rd glutamic acid of SEQ ID NO: 6 is lysine,
The 297th glutamine of SEQ ID NO: 6 is lysine,
301st histidine of SEQ ID NO: 6 to lysine
The 304th proline of SEQ ID NO: 6 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: 7, and 60 substitutions occurred in this region. (Japanese Patent Laid-Open No. 2011-206046, Patent Document 2).
(1) A polynucleotide encoding the Fc-binding protein FcRm68 consisting of the amino acid sequence shown in SEQ ID NO: 8, and using the polynucleotide consisting of the sequence shown in SEQ ID NO: 9 as a template, SEQ ID NO: 10 (5′-CATGCCATGGGACAAGTAGATATCCCCCAAGCTGTGGATAAGCTGCCAACC- PCR was carried out using the oligonucleotides consisting of the sequences described in 3 ′) and SEQ ID NO: 11 (5′-CCCGGAAGCTTAGCCCGCAGCTCCGGGGTCTTCGTTTGTTACCCAGTAC-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 the Fc binding protein 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に記載の発現ベクターｐＴｒｃｐｅｌにライゲーションし、これを用いて大腸菌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を添加した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 to the expression vector pTrcpel described in Example 1 previously digested with restriction enzymes NcoI and HindIII. E. coli strain W3110 was transformed.
(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. pTrcFcRm68-CG 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: 12, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 13, respectively. In SEQ ID NO: 12, the amino acid sequence of the PelB signal peptide is from the first methionine to the 22nd alanine, and the amino acid sequence of the Fc-binding protein FcRm68 is from the 25th glutamine to the 298th aspartic acid. Up to the 300th glycine is the amino acid sequence of the cysteine tag. In SEQ ID NO: 12, 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: 7 from the 16th glutamine to the 289th position. It corresponds to the area up to valine.

Example 2 Preparation of Fc-binding protein expression vector to which oligopeptide of the present invention was added (Part 1)
Coding a polypeptide (FcRm68-T1) in which an oligopeptide consisting of the amino acid sequence shown in SEQ ID NO: 14 (amino acid sequence: CTRCG) is added to the C-terminal side of the Fc binding protein FcRm68 consisting of the amino acid sequence shown in SEQ ID NO: 8 The FcRm68-T1 expression vector was prepared by inserting the polynucleotide to be inserted into the expression vector pTrcpel prepared in Example 1.
(1) Using the expression vector pTrcFcRm68-CG prepared in Comparative Example 1 as a template, PCR consisting of an oligonucleotide having the sequence described in SEQ ID NO: 10 and SEQ ID NO: 15 (5′-CCCAAGCTTTCCGCAACGGGTGCAGCTCCGGGGTCTTCTGTTGTTTCCCCAGTACTTTTCAACTCAAG-3 ′) did. 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 55 ° C. for 5 seconds, and a third step at 72 ° C. for 1 minute. The reaction for 1 cycle was repeated 30 times. The obtained PCR product was purified to obtain a polynucleotide encoding the Fc-binding protein FcRm68-T1.
(2) The polynucleotide encoding FcRm68-T1 was digested with restriction enzymes NcoI and HindIII and then ligated to the expression vector pTrcpel prepared in Example 1 previously digested with restriction enzymes NcoI and HindIII, and E. coli strain W3110 was used. Was transformed.
(3) The obtained transformant is cultured in an LB medium supplemented with 100 μg / mL carbenicillin sodium, and the expression vector is extracted to thereby express an expression vector pTrcFcRm68 containing a polynucleotide encoding the Fc-binding protein FcRm68-T1. -T1 was obtained.
(4) The nucleotide sequence was analyzed in the same manner as in Example 1 (4).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcFcRm68-T1 is shown in SEQ ID NO: 16, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 17, respectively. In SEQ ID NO: 16, the amino acid sequence of the PelB signal peptide is from the first methionine to the 22nd alanine, and the amino acid sequence of the Fc-binding protein FcRm68 is from the 25th glutamine to the 298th aspartic acid. Up to the 303rd glycine is the amino acid sequence of the oligopeptide described in SEQ ID NO: 14.

Example 3 Preparation of an Fc-binding protein expression vector to which the oligopeptide of the present invention was added (part 2)
The amino acid sequence described in SEQ ID NO: 8 in the same manner as in Example 2 except that an oligonucleotide consisting of the sequence described in SEQ ID NO: 10 and SEQ ID NO: 19 (5′-CCCAAGCTTATCCGCAGGTTTTTGCAGTCCGGGGTCTTTCGTTTGTTTCCCCAGTACTTTCAACTCAAG-3 ′) was used as a PCR primer. A polynucleotide encoding a polypeptide (FcRm68-T2) obtained by adding an oligopeptide (amino acid sequence: CKTCG) consisting of the amino acid sequence shown in SEQ ID NO: 18 to the C-terminal side of the Fc-binding protein FcRm68 consisting of The FcRm68-T2 expression vector pTrcFcRm68-T2 was prepared by inserting it into the expression vector pTrcpel prepared in step 1. Thereafter, the nucleotide sequence was analyzed by the method described in Example 1 (4).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcFcRm68-T2 is shown in SEQ ID NO: 20, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 21, respectively. In SEQ ID NO: 20, the amino acid sequence of the PelB signal peptide is from the first methionine to the 22nd alanine, and the amino acid sequence of the Fc binding protein FcRm68 is from the 25th glutamine to the 298th aspartic acid. Up to the 303rd glycine is the amino acid sequence of the oligopeptide described in SEQ ID NO: 18.

Example 4 Preparation of Fc-binding protein expression vector to which oligopeptide of the present invention was added (part 3)
SEQ ID NO: 8 described in SEQ ID NO: 8 in the same manner as in SEQ ID NO: 8, except that the oligonucleotide consisting of the sequence described in SEQ ID NO: 10 and SEQ ID NO: 23 (5′-CCCAAGCTTATCCCCAGGGTATCGGTTGCGGCAGTCCCGGGTCCTTCTGTTGTTTACCCAGTACTTTCAACTCAAG-3 ′) A polynucleotide encoding a polypeptide (FcRm68-T3) in which an oligopeptide consisting of the amino acid sequence shown in SEQ ID NO: 22 (amino acid sequence: CRNDTCG) was added to the C-terminal side of the Fc-binding protein FcRm68 consisting of: This was inserted into the expression vector pTrcpel prepared in (1) to prepare the FcRm68-T3 expression vector pTrcFcRm68-T3. Thereafter, the nucleotide sequence was analyzed by the method described in Example 1 (4).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcFcRm68-T3 is shown in SEQ ID NO: 24, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 25, respectively. In SEQ ID NO: 24, the amino acid sequence of the PelB signal peptide is from the first methionine to the 22nd alanine, and the amino acid sequence of the Fc binding protein FcRm68 is from the 25th glutamine to the 298th aspartic acid. Up to the 305th glycine is the amino acid sequence of the oligopeptide described in SEQ ID NO: 22.

Example 5 Preparation of Fc-binding protein expression vector to which oligopeptide of the present invention was added (Part 4)
SEQ ID NO: 8 described in SEQ ID NO: 8 in the same manner as in SEQ ID NO: 8, except that the oligonucleotide consisting of the sequence described in SEQ ID NO: 10 and SEQ ID NO: 27 (5′-CCCAAGCTTATCCCCACGGATCGCTCCCGGCATCTCGGGGTCTTTCTGTTGTTACCCAGACTTTCAACTCAAG-3 ′) was used as a PCR primer. A polynucleotide encoding a polypeptide (FcRm68-T4) in which an oligopeptide consisting of the amino acid sequence shown in SEQ ID NO: 26 (amino acid sequence: CRSDPCG) was added to the C-terminal side of the Fc-binding protein FcRm68 consisting of: The FcRm68-T4 expression vector pTrcFcRm68-T4 was prepared by inserting it into the expression vector pTrcpel prepared in step 1. Thereafter, the nucleotide sequence was analyzed by the method described in Example 1 (4).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcFcRm68-T4 is shown in SEQ ID NO: 28, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 29, respectively. In SEQ ID NO: 28, the amino acid sequence of the PelB signal peptide is from the first methionine to the 22nd alanine, and the amino acid sequence of the Fc binding protein FcRm68 is from the 25th glutamine to the 298th aspartic acid from the 299th cysteine. Up to the 305th glycine is the amino acid sequence of the oligopeptide described in SEQ ID NO: 26.

Example 6 Preparation of an Fc-binding protein expression vector to which the oligopeptide of the present invention was added (part 5)
SEQ ID NO: 8 described in SEQ ID NO: 8 in the same manner as in SEQ ID NO: 8, except that an oligonucleotide consisting of the sequence described in SEQ ID NO: 10 and SEQ ID NO: 31 (5′-CCCAAGCTTATCCCCACACGCTTTTCGCCCAGGCAGTCCCGGGGTCTTCTGTTGTTTACCCAGACTACTTCAACTCAAG-3 ′) was used as a PCR primer. A polynucleotide encoding a polypeptide (FcRm68-T5) obtained by adding an oligopeptide (amino acid sequence: CLGESVCG) consisting of the amino acid sequence shown in SEQ ID NO: 30 to the C-terminal side of the Fc binding protein FcRm68 consisting of Was inserted into the expression vector pTrcpel prepared in step 1 to prepare the FcRm68-T5 expression vector pTrcFcRm68-T5. Thereafter, the nucleotide sequence was analyzed by the method described in Example 1 (4).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcFcRm68-T5 is shown in SEQ ID NO: 32, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 33, respectively. In SEQ ID NO: 32, the amino acid sequence of the PelB signal peptide is from the first methionine to the 22nd alanine, and the amino acid sequence of the Fc binding protein FcRm68 is from the 25th glutamine to the 298th aspartic acid. Up to the 306th glycine is the amino acid sequence of the oligopeptide described in SEQ ID NO: 30.

Example 7 Preparation of Fc-binding protein expression vector added with oligopeptide of the present invention (No. 6)
Except that the oligonucleotide consisting of the sequence described in SEQ ID NO: 10 and SEQ ID NO: 35 (5′-CCCAAGCTTATCCCCACGGGTTCGCCTTCAATGCAGTCCCGGGGTCTTCTGTTGTTTACCCAGTACTTTCAACTCAAG-3 ′) was used as a PCR primer in the same manner as in SEQ ID NO: 8, A polynucleotide encoding a polypeptide (FcRm68-T6) obtained by adding an oligopeptide (amino acid sequence: CIEGEPCG) consisting of the amino acid sequence shown in SEQ ID NO: 34 to the C-terminal side of the Fc-binding protein FcRm68 consisting of: Was inserted into the expression vector pTrcpel prepared in step 1 to prepare the FcRm68-T6 expression vector pTrcFcRm68-T6. Thereafter, the nucleotide sequence was analyzed by the method described in Example 1 (4).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcFcRm68-T6 is shown in SEQ ID NO: 36, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 37, respectively. In SEQ ID NO: 36, the amino acid sequence of the PelB signal peptide is from the first methionine to the 22nd alanine, and the amino acid sequence of the Fc binding protein FcRm68 is from the 25th glutamine to the 298th aspartic acid from the 299th cysteine. Up to the 306th glycine is the amino acid sequence of the oligopeptide described in SEQ ID NO: 34.

Example 8 Preparation of Fc-binding protein expression vector to which oligopeptide of the present invention was added (part 7)
SEQ ID NO: 8 described in SEQ ID NO: 8 in the same manner as in SEQ ID NO: 8, except that an oligonucleotide consisting of the sequence described in SEQ ID NO: 10 and SEQ ID NO: 39 (5′-CCCAAGCTTATCCCCATTTGCCCGTTATGGCAGTCCCGGGGTCTTCTGTTGTTTACCCAGACTACTCAACTCAAG-3 ′) was used as a PCR primer. A polynucleotide encoding a polypeptide (FcRm68-T7) obtained by adding an oligopeptide (amino acid sequence: CHNGKCG) consisting of the amino acid sequence shown in SEQ ID NO: 38 to the C-terminal side of the Fc-binding protein FcRm68 consisting of: The FcRm68-T7 expression vector pTrcFcRm68-T7 was prepared by inserting it into the expression vector pTrcpel prepared in step 1. Thereafter, the nucleotide sequence was analyzed by the method described in Example 1 (4).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcFcRm68-T7 is shown in SEQ ID NO: 40, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 41, respectively. In SEQ ID NO: 40, the amino acid sequence of the PelB signal peptide is from the first methionine to the 22nd alanine, and the amino acid sequence of the Fc-binding protein FcRm68 is from the 25th glutamine to the 298th aspartic acid. Up to the 305th glycine is the amino acid sequence of the oligopeptide described in SEQ ID NO: 38.

Example 9 Preparation of Fc-binding protein expression vector added with oligopeptide of the present invention (No. 8)
The amino acid sequence described in SEQ ID NO: 8 is the same as that in SEQ ID NO: 8, except that an oligonucleotide consisting of the sequence described in SEQ ID NO: 10 and SEQ ID NO: 43 (5′-CCCAAGCTTATCCCCATTTGCCCGCTGCCGCAGTCCCGGGGTCTTTCTGTTGTTACCCAGACTTTCAACTC-3 ′) was used as a PCR primer. A polynucleotide encoding a polypeptide (FcRm68-T8) in which an oligopeptide consisting of the amino acid sequence shown in SEQ ID NO: 42 (amino acid sequence: CGSGKCG) was added to the C-terminal side of the Fc-binding protein FcRm68 consisting of: The FcRm68-T8 expression vector pTrcFcRm68-T8 was prepared by inserting it into the expression vector pTrcpel prepared in step 1. Thereafter, the nucleotide sequence was analyzed by the method described in Example 1 (4).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcFcRm68-T8 is shown in SEQ ID NO: 44, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 45, respectively. In SEQ ID NO: 44, the amino acid sequence of the PelB signal peptide is from the first methionine to the 22nd alanine, and the amino acid sequence of the Fc binding protein FcRm68 is from the 25th glutamine to the 298th aspartic acid. Up to the 305th glycine is the amino acid sequence of the oligopeptide described in SEQ ID NO: 42.

Example 10 Preparation of Fc-binding protein expression vector added with oligopeptide of the present invention (No. 9)
SEQ ID NO: 8 described in SEQ ID NO: 8 in the same manner as in SEQ ID NO: 8, except that an oligonucleotide consisting of the sequence described in SEQ ID NO: 10 and SEQ ID NO: 47 (5′-CCCAAGCTTATCCCCATTTGCCCGCCATCGCAGTCCCGGGTCTTTCTGTTGTTACCCAGTACTTTCACTACT-3 ′) was used as a PCR primer. A polynucleotide encoding a polypeptide (FcRm68-T9) obtained by adding an oligopeptide (amino acid sequence: CDGGKCG) consisting of the amino acid sequence shown in SEQ ID NO: 46 to the C-terminal side of the Fc-binding protein FcRm68 consisting of: The FcRm68-T9 expression vector pTrcFcRm68-T9 was prepared by inserting it into the expression vector pTrcpel prepared in step 1. Thereafter, the nucleotide sequence was analyzed by the method described in Example 1 (4).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcFcRm68-T9 is shown in SEQ ID NO: 48, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 49, respectively. In SEQ ID NO: 48, the amino acid sequence of the PelB signal peptide is from the first methionine to the 22nd alanine, and the amino acid sequence of the Fc binding protein FcRm68 is from the 25th glutamine to the 298th aspartic acid. Up to the 305th glycine is the amino acid sequence of the oligopeptide described in SEQ ID NO: 46.

Example 11 Evaluation of Fc Binding Protein Productivity (Part 1)
(1) E. coli strain W3110 was transformed with the expression vectors prepared in Comparative Example 1 and Examples 2 to 10, and inoculated into 2YT liquid medium supplemented with 100 μg / mL carbenicillin sodium, and then aerobic at 37 ° C. overnight. Pre-culture was performed by shaking culture.
(2) 200 μL of the preculture was inoculated into 20 mL of 2YT liquid medium supplemented with 100 μg / mL of carbenicillin sodium, and cultured with shaking at 37 ° C. aerobically.
(3) 1.5 hours after the start of the culture, change the culture temperature to 20 ° C., shake culture for 30 minutes, add IPTG to a final concentration of 0.01 mM, and continue aerobic at 20 ° C. overnight. Cultured with shaking.
(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) is measured for antibody binding activity using the ELISA method described below, and compared with the value in a known concentration of Fc binding protein. 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 2% (w / v) SKIM MILK (manufactured by BD).
(5-2) After washing with a washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% (w / v) Tween 20 and 150 mM NaCl), the prepared Fc-binding protein was The containing solution was reacted with immobilized gamma globulin (1 hour at 30 ° C.).
(5-3) After completion of the reaction, an anti-FcγRI antibody (manufactured by R & D Systems) that was washed with the washing buffer and diluted to 50 ng / mL was added at 100 μL / well.
(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 at.
(5-5) 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.
(6) The concentration of Fc-binding protein after culture was measured from a calibration curve using purified Fc-binding protein of known concentration by ELISA, and after incubation at 600 nm absorbance measured as the amount of cells at the end of the culture The productivity of Fc binding protein per cell was calculated by dividing the concentration of Fc binding protein.

  The results are shown in Table 2. Using the expression vectors prepared in Examples 2 to 10 as Fc-binding protein expression vectors, it was confirmed that the productivity was improved as compared with the expression vector prepared in Comparative Example 1. That is, it can be seen that the productivity of the protein is improved by adding the oligopeptide of the present invention to the protein.

実施例１２ Ｆｃ結合性タンパク質の調製
（１）比較例１（ｐＴｒｃＦｃＲｍ６８−ＣＧ）、実施例４（ｐＴｒｃＦｃＲｍ６８−Ｔ３）および実施例８（ｐＴｒｃＦｃＲｍ６８−Ｔ７）に記載の発現ベクターにより形質転換された大腸菌Ｗ３１１０株を１００μｇ／ｍＬのアンピシリンナトリウムを添加した２×ＹＴ液体培地１００ｍＬに接種し、５００ｍＬフラスコにて３０℃で一晩振とう培養し、前培養液を調製した。
（２）３５ｇ／Ｌのグリセロール、５０ｇ／Ｌの酵母エキス、３ｇ／Ｌのリン酸三ナトリウム十二水和物、９ｇ／Ｌのリン酸水素二ナトリウム十二水和物、１ｇ／Ｌの塩化アンモニウム、および１００ｍｇ／Ｌのアンピシリンナトリウムを含む０．６Ｌの液体培地に、（１）の前培養液３０ｍＬを接種後、１Ｌ発酵槽を用いて、培養液温度３０℃、ｐＨ６．９から７．１、通気量１ＶＶＭ、溶存酸素濃度３０％飽和濃度の条件に設定し、培養を開始した。なおｐＨ制御には、酸として５０％リン酸、アルカリとして１４％アンモニア水を、それぞれ使用し、溶存酸素の制御は撹拌速度を変化させることで行ない、撹拌回転数は下限５００ｒｐｍ、上限１０００ｒｐｍに設定した。
（３）培養開始から約５時間後に温度を２５℃に下げ、設定温度に到達したことを確認した後、終濃度が０．５ｍＭになるようＩＰＴＧ（イソプロピル−β−チオガラクトピラノシド）を添加し、引き続き２５℃で培養を継続した。
（４）培養開始から約２９時間後に培養を停止し、培養液を８０００ｒｐｍ、１０分間の遠心分離により菌体を回収した。
（５）回収した菌体を０．１５Ｍの塩化ナトリウムを含む２０ｍＭのリン酸緩衝液（ｐＨ７．０）６０ｍＬに懸濁し、菌体を破砕した。菌体破砕は超音波発生装置（インソネーター２０１Ｍ（商品名）、久保田商事製）を用いて、４℃で約５分間、約１７０Ｗの出力で処理した。
（６）破砕処理液を４℃、１５分間、１２０００ｒｐｍの遠心分離を２回行ない、上清を回収した。
（７）回収した破砕処理液の上清を、１０ｍＬゲル量のＩｇＧセファロースカラム（ＧＥヘルスケア社製）にアプライし、１５０ｍＭの塩化ナトリウムおよび０．０５％のＴｗｅｅｎ２０を含む２０ｍＭのトリス塩酸緩衝液（ｐＨ７．４）で洗浄後、０．０５％のＴｗｅｅｎ２０を含む２０ｍＭのクエン酸緩衝液（ｐＨ３．０）でＦｃ結合性タンパク質を含む画分を溶出した。溶出した画分に含まれるＦｃ結合性タンパク質の純度をＳＤＳポリアクリルアミド電気泳動により確認した結果、単一バンドであることを確認した。
（８）ＩｇＧセファロースカラムで精製したＦｃ結合性タンパク質をＡｍｉｃｏｎ Ｕｌｔｒａ−１０Ｋ（ミリポア社製）により濃縮後、０．２Ｍの硫酸ナトリウムと１ｍＭのＥＤＴＡと０．２ｍＭの〔２−ｃａｒｂｏｘｙｅｔｈｙｌ〕ｐｈｏｓｐｈｉｎｅｈｙｄｒｏｃｈｌｏｒｉｄｅ（ＴＣＥＰ）とを含んだ２０ｍＭのＭＥＳ（ｐＨ５．５）への緩衝液交換を行なった。
（９）濃縮したＦｃ結合性タンパク質を含む画分を２８０ｎｍの吸光度にて測定し、Ｆｃ結合性タンパク質として１０ｍｇ／ｍＬとなるよう濃度調製した。

Example 12 Preparation of Fc-binding protein (1) E. coli W3110 transformed with the expression vector described in Comparative Example 1 (pTrcFcRm68-CG), Example 4 (pTrcFcRm68-T3) and Example 8 (pTrcFcRm68-T7) The strain was inoculated into 100 mL of 2 × YT liquid medium supplemented with 100 μg / mL ampicillin sodium, and cultured with shaking overnight at 30 ° C. in a 500 mL flask to prepare a preculture solution.
(2) 35 g / L glycerol, 50 g / L yeast extract, 3 g / L trisodium phosphate dodecahydrate, 9 g / L disodium hydrogen phosphate dodecahydrate, 1 g / L chloride A 0.6 L liquid medium containing ammonium and 100 mg / L ampicillin sodium was inoculated with 30 mL of the pre-culture solution of (1), and then the culture solution temperature was 30 ° C. and pH 6.9 to 7. The culture was started after setting the conditions of 1, aeration volume of 1 VVM, and a dissolved oxygen concentration of 30% saturation. For pH control, 50% phosphoric acid as acid and 14% ammonia water as alkali are used, respectively, and dissolved oxygen is controlled by changing the stirring speed. The stirring speed is set at a lower limit of 500 rpm and an upper limit of 1000 rpm. did.
(3) After about 5 hours from the start of the culture, the temperature was lowered to 25 ° C., and after confirming that the set temperature was reached, IPTG (isopropyl-β-thiogalactopyranoside) was added so that the final concentration was 0.5 mM. Then, the culture was continued at 25 ° C.
(4) The culture was stopped about 29 hours after the start of the culture, and the cells were collected by centrifugation at 8000 rpm for 10 minutes.
(5) The collected cells were suspended in 60 mL of 20 mM phosphate buffer (pH 7.0) containing 0.15 M sodium chloride, and the cells were crushed. The microbial cell disruption was processed with an output of about 170 W at 4 ° C. for about 5 minutes using an ultrasonic generator (Insonator 201M (trade name), manufactured by Kubota Corporation).
(6) The crushing solution was centrifuged twice at 12000 rpm at 4 ° C. for 15 minutes, and the supernatant was collected.
(7) The supernatant of the collected crushing solution is applied to a 10 mL gel amount of IgG Sepharose column (GE Healthcare) and 20 mM Tris-HCl buffer containing 150 mM sodium chloride and 0.05% Tween 20 After washing with (pH 7.4), the fraction containing Fc-binding protein was eluted with 20 mM citrate buffer (pH 3.0) containing 0.05% Tween20. As a result of confirming the purity of the Fc binding protein contained in the eluted fraction by SDS polyacrylamide electrophoresis, it was confirmed to be a single band.
(8) Fc-binding protein purified on an IgG sepharose column was concentrated with Amicon Ultra-10K (Millipore), 0.2M sodium sulfate, 1mM EDTA, and 0.2mM [2-carbethylethyl] phosphine hydrochloride (TCEP). ) Was added to 20 mM MES (pH 5.5).
(9) The fraction containing the concentrated Fc binding protein was measured at an absorbance of 280 nm, and the concentration was adjusted to 10 mg / mL as the Fc binding protein.

Example 13 Evaluation of Immobilization Rate of Fc Binding Protein to Insoluble Carrier (1) 1,2-ethanediol diglycidyl ether, trisaminoethylamine, and iodoacetic acid were sequentially added to the hydroxyl group of Toyopearl (manufactured by Tosoh Corporation) as an insoluble carrier. By reacting, iodoacetamidotoyopearl gel was prepared.
(2) 10 mg / mL solution of each Fc-binding protein (FcRm68-CG, FcRm68-T3 and FcRm68-T7) prepared in Example 12 and 1M Tris buffer (pH 8.5) are shown in Table 3 respectively. The reaction solution was prepared by mixing at the ratio shown in FIG.

（３）（１）で作製したヨードアセトアミドトヨパールゲル３０μＬをスピンカラム（バイオラッド社製）に充填したカラムに（２）で調製した反応液をアプライし、２５℃で２時間反応させることでＦｃ結合性タンパク質をヨードアセトアミドトヨパールゲルに固定化した。
（３）反応終了後、遠心操作により反応残渣を回収し、５０ｍＭのクエン酸緩衝液（ｐＨ３．０）にて洗浄することで、Ｆｃ結合性タンパク質固定化ゲルを調製した。
（４）反応残渣及び洗浄のクエン酸緩衝液に含まれるタンパク質量を、吸光度計（日立製作所製）で測定した２８０ｎｍの吸収度から算出し、算出したタンパク質量を反応に供したタンパク質量から差し引くことで、ヨードアセトアミドトヨパールへのＦｃ結合性タンパク質固定化量を計算した。また、ＦｃＲｍ６８−ＣＧ、ＦｃＲｍ６８−Ｔ３およびＦｃＲｍ６８−Ｔ７の固定化されたタンパク質量を反応に供したタンパク質量で除することで固定化率を算出した。

(3) The reaction solution prepared in (2) was applied to a column filled with 30 μL of iodoacetamidotoyopearl gel prepared in (1) in a spin column (Bio-Rad), and reacted at 25 ° C. for 2 hours. Fc binding protein was immobilized on iodoacetamido toyopearl gel.
(3) After completion of the reaction, the reaction residue was collected by centrifugation and washed with 50 mM citrate buffer (pH 3.0) to prepare an Fc-binding protein-immobilized gel.
(4) The amount of protein contained in the reaction residue and washing citrate buffer is calculated from the absorbance at 280 nm measured with an absorptiometer (manufactured by Hitachi, Ltd.), and the calculated amount of protein is subtracted from the amount of protein subjected to the reaction. Thus, the amount of Fc-binding protein immobilized on iodoacetamidotoyopearl was calculated. Moreover, the immobilization rate was calculated by dividing the amount of protein immobilized on FcRm68-CG, FcRm68-T3 and FcRm68-T7 by the amount of protein subjected to the reaction.

  As a result, FcRm68-CG (Comparative Example 1) was immobilized at 20.1 mg per mL of gel, and the immobilization rate was 67%. On the other hand, 19.3 mg of FcRm68-T3 (Example 4) was immobilized per mL of gel, and the immobilization rate was 96%. Further, 19.1 mg of FcRm68-T7 (Example 8) was immobilized per mL of the gel, and the immobilization rate was 95%. A summary of the immobilization rate results is shown in FIG. From the above results, it can be seen that the immobilization rate is improved by adding the oligopeptide of the present invention to a protein.

Example 14 Performance Evaluation of Fc Binding Protein Immobilized Gel (1) The Fc binding protein immobilized gel prepared in Example 13 was packed in a spin column (Bio-Rad).
(2) Human polyclonal antibody (gamma globulin preparation, chemical and serum) diluted with 50 mM phosphate buffer (pH 7.0) after equilibrating the gel by adding 150 μL of 50 mM phosphate buffer (pH 7.0) (Manufactured by Therapeutic Research Institute) was added to 10 mg and shaken at room temperature for 120 minutes.
(3) After eluting unadsorbed human antibody from the column by washing thoroughly with 50 mM phosphate buffer containing 150 mM NaCl, 100 mM citrate buffer (pH 3.0) is added to the column. The human antibody adsorbed on the gel was eluted.
(4) The antibody concentration in the eluate was calculated by measuring the absorbance at 280 nm. The absorbance at 280 nm (optical path length: 10 mm) of the 1 mg / ml antibody solution is calculated as 1.4.

  The results of calculating the amount of antibody adsorbed per mL of each gel are shown in FIG. Since FcRm68-CG (Comparative Example 1), FcRm68-T3 (Example 4), and FcRm68-T7 (Example 8) all show the same amount of antibody adsorption, the oligopeptide of the present invention is used. It was confirmed that even when immobilized on an insoluble carrier, the performance as an antibody (immunoglobulin) adsorbent was not affected.

Comparative Example 2 Preparation of Fc-binding protein expression vector to which cysteine tag was added (part 2)
A polynucleotide encoding a polypeptide (FcRm72c-CG) in which a cysteine tag (amino acid sequence: CG) is added to the C-terminal side of the Fc binding protein FcRm72c consisting of the amino acid sequence set forth in SEQ ID NO: 50 is prepared in Example 1. An FcRm72c-CG expression vector was prepared by inserting the expression vector pTrcpel. FcRm72c is an amino acid sequence of the Fc binding protein FcRm68 described in SEQ ID NO: 8,
49th glutamine of SEQ ID NO: 8 is proline,
144th asparagine of SEQ ID NO: 8 is aspartic acid,
180th threonine of SEQ ID NO: 8 is serine,
The 209th glutamine of SEQ ID NO: 8 is arginine,
The 238th glutamic acid of SEQ ID NO: 8 is changed to aspartic acid,
Each is an Fc binding protein with amino acid substitution.
(1) A polynucleotide encoding the Fc-binding protein FcRm72c consisting of the amino acid sequence shown in SEQ ID NO: 50, which is a polynucleotide consisting of the sequence shown in SEQ ID NO: 51 as a template, and described in SEQ ID NO: 10 and SEQ ID NO: 11. PCR was performed using the oligonucleotide of No. 1 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 Fc binding protein FcRm72c-CG was obtained.
(2) The obtained polynucleotide encoding FcRm72c-CG was digested with restriction enzymes NcoI and HindIII, and then ligated to the expression vector pTrcpel described in Example 1 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 FcRm72c-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. pTrcFcRm72c-CG was extracted.
Of the expression vector pTrcFcRm72c-CG produced in (4), the nucleotide sequence of the polynucleotide encoding FcRm72c and the surrounding region was analyzed in the same manner as in Example 1 (4).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcFcRm72c-CG is shown in SEQ ID NO: 52, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 53, respectively. In SEQ ID NO: 52, the first methionine to the 22nd alanine are the PelB signal peptide, the 25th glutamine to the 298th aspartic acid are the amino acid sequence of the Fc binding protein FcRm72c, the 299th cysteine to the 300th Up to glycine is a cysteine tag. In SEQ ID NO: 52, the amino acid sequence of FcRm72c (region from the 25th glutamine to the 298th aspartic acid) is the 289th position from the 16th glutamine in the amino acid sequence of the human Fc receptor FcγRI described in SEQ ID NO: 7. It corresponds to the area up to valine.

Example 15 Preparation of Fc-binding protein expression vector added with oligopeptide of the present invention (No. 10)
Except that the expression vector pTrcFcRm72c-CG prepared in Comparative Example 2 was used as a template and an oligonucleotide consisting of the sequences described in SEQ ID NO: 10 and SEQ ID NO: 23 was used as a PCR primer, A polypeptide encoding a polypeptide (FcRm72c-T3) in which an oligopeptide (amino acid sequence: CRNDTCG) consisting of the amino acid sequence shown in SEQ ID NO: 22 is added to the C-terminal side of the Fc binding protein FcRm72c consisting of the amino acid sequence shown in 50 Nucleotides were inserted into the expression vector pTrcpel prepared in Example 1 to prepare an FcRm72c-T3 expression vector pTrcFcRm72c-T3. Thereafter, the nucleotide sequence was analyzed by the method described in Example 1 (4).

  The amino acid sequence of the polypeptide expressed by the expression vector pTrcFcRm72c-T3 is shown in SEQ ID NO: 54, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 55, respectively. In SEQ ID NO: 54, the amino acid sequence of the PelB signal peptide is from the first methionine to the 22nd alanine, and the amino acid sequence of the Fc-binding protein FcRm72c is from the 25th glutamine to the 298th aspartic acid from the 299th cysteine. Up to the 305th glycine is the amino acid sequence of the oligopeptide described in SEQ ID NO: 22.

Example 16 Evaluation of Fc Binding Protein Productivity (Part 2)
After transforming E. coli strain W3110 with the expression vectors described in Comparative Example 2 (pTrcFcRm72c-CG) and Example 15 (pTrcFcRm72c-T3), the productivity of Fc-binding protein was evaluated in the same manner as in Example 11. .

  The results are shown in Table 4. By using the expression vector prepared in Example 15 as an Fc-binding protein expression vector, it was confirmed that productivity was improved as compared with the expression vector prepared in Comparative Example 2. That is, similar to the result of Example 11, it can be seen that the productivity of the protein is improved by adding the oligopeptide of the present invention to the protein.

  By adding the oligopeptide of the present invention to the N-terminal side or C-terminal side of the protein, the protein can be efficiently immobilized on the surface of the insoluble carrier. Therefore, it can be used for an adsorbent, a sensor chip or the like using an immobilized protein. The oligopeptide of the present invention is also useful for improving the productivity of the protein.

Claims (10)

An oligopeptide consisting of 5 to 8 amino acid residues for immobilizing a protein on an insoluble carrier,
At least, an amino acid residue, the N-terminal side cysteine, 2 amino acid residues are cysteine C-terminal - glycine der is, and SEQ ID NO: 14,18,22,26,30,34,38,42,46 ing the amino acid sequence of any one of the oligopeptide. An oligopeptide-added protein comprising the oligopeptide according to claim 1 added to the N-terminal side or C-terminal side of the protein. The oligopeptide-added protein according to claim 2 , wherein the protein is an Fc-binding protein. The oligopeptide-added protein according to claim 3 , comprising the amino acid sequence of any one of SEQ ID NOs: 16, 20, 24, 28, 32, 36, 40, 44, 48, and 54. A protein adsorbent obtained by immobilizing the oligopeptide-added protein according to any one of claims 2 to 4 on an insoluble carrier. A protein purification method using the adsorbent according to claim 5 . A polynucleotide encoding the oligopeptide-added protein according to any one of claims 2 to 4 . An expression vector comprising the polynucleotide according to claim 7 . A transformant obtained by transforming a host with the expression vector according to claim 8 . A method for producing an oligopeptide-added protein, comprising culturing the transformant according to claim 9 and recovering the oligopeptide-added protein from the obtained culture solution.

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