Method for producing Fc-binding proteins using genetically modified E. coli

Abstract

The objective is to provide a method for efficiently producing the Fc-binding protein using genetically modified Escherichia coli capable of expressing the aforementioned protein. [Solution] The present invention provides a method for producing an Fc-binding protein, comprising the steps of culturing genetically modified Escherichia coli containing a polynucleotide encoding an Fc-binding protein and expressing the protein, and recovering the protein, wherein the genetically modified Escherichia coli is strain JM109 containing a polynucleotide encoding an Fc-binding protein, and the culture medium used for culturing the genetically modified Escherichia coli contains at least peptone and yeast extract at a concentration of 10 g / L or more, thereby solving the above problem.

Description

Technical Field

[0001] The present invention relates to a method for efficiently industrially producing a protein using a genetically engineered Escherichia coli capable of expressing an Fc-binding protein obtained by genetic engineering techniques. In particular, the present invention relates to a particularly efficient production method when the Fc-binding protein is a human neonatal Fc receptor (FcRn).

Background Art

[0002] An Fc-binding protein (Fc receptor) is a receptor protein that binds to the Fc region of an immunoglobulin molecule, binds to an immune complex of an antigen and an immunoglobulin, and conducts signal transduction into cells (Non-Patent Document 1). Each molecule recognizes a single or the same group of immunoglobulin isotypes by a recognition domain belonging to the immunoglobulin superfamily on the recognition domain of the Fc receptor. This determines which accessory cells are activated in the immune response.

[0003] Fc receptors can be further classified into several subtypes, including Fcγ receptors, which are receptors for immunoglobulin G (IgG), Fcα receptors, Fcε receptors, etc. (Non-Patent Documents 1 and 2). Among them, the human neonatal Fc receptor (FcRn) is a major histocompatibility complex (MHC) class I-related molecule different from the human Fcγ receptor belonging to the immunoglobulin superfamily, and is composed of a heavy chain (α chain) and β2-microglobulin (β chain) (Non-Patent Document 3).

[0004] The antibody adsorption ability of such Fc-binding proteins can also be used as a protein responsible for the capture function of various antibody purification chromatography gels. Therefore, production methods using recombinant organisms capable of expressing Fc-binding proteins have been studied so far.

[0005] For example, examples of using HEK293 cells as a host for the production of Fc-binding proteins are disclosed (Patent Document 1), examples of using Sf9 cells as a host (Non-Patent Document 4), and examples of using yeast as a host (Non-Patent Document 5).

[0006] From the perspective of producing proteins in large quantities and inexpensively on a commercial scale, using Escherichia coli as a host is considered the most suitable method. As an example of using Escherichia coli as a host, Non-Patent Document 6 discloses a method for producing Fc-binding proteins using the BL21 derivative Rosetta-gami 2 strain as a host. Non-Patent Document 7 also discloses a method for producing Fc-binding proteins using BL21(DE3) as a host. Although several methods for producing Fc-binding proteins using Escherichia coli as a host have been investigated, there has been a need for further improvements in production efficiency. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2017-207494 [Non-patent literature]

[0008] [Non-Patent Document 1] Takai T.,Jpn.J.Clin.Immunol.,28,318-326,2005 [Non-Patent Document 2] J.Galon et al.,Eur.J.Immunol.,27,1928-1932,1997 [Non-Patent Document 3] NESimister et al.,Nature,337,184-187,1989 [Non-Patent Document 4] B. Szikora et al.,PLoS One,12,e0185662,2017. [Non-Patent Document 5] CHLee et al.,Protein Expr.Purif.,71,42-48,2010. [Non-Patent Document 6] WKNg et al.,Protein Expr.Purif.,127,73-80,2016. [Non-Patent Document 7] JTA Andersen et al., J. Immunol. Methods, 331, 39-49, 2008. [Overview of the project] [Problems that the invention aims to solve]

[0009] The object of the present invention is to provide a method for efficiently producing an Fc-binding protein using genetically modified Escherichia coli capable of expressing the Fc-binding protein. [Means for solving the problem]

[0010] The inventors have found suitable culture conditions for genetically modified Escherichia coli capable of expressing Fc-binding proteins, which are effective for the efficient production of the Fc-binding protein.

[0011] In other words, the present invention encompasses the following aspects: [1] A method for producing an Fc-binding protein, comprising the steps of culturing genetically modified Escherichia coli containing a polynucleotide encoding an Fc-binding protein and expressing the protein, and recovering the protein, The method for producing the genetically modified Escherichia coli, wherein the genetically modified Escherichia coli is strain JM109 containing a polynucleotide encoding an Fc-binding protein, and the culture medium used for culturing the genetically modified Escherichia coli contains at least peptone and 10 g / L or more of yeast extract. [2] The method for producing the peptone according to [1], wherein the peptone is derived from any of soybeans, cotton, broad beans, lupin beans, corn, potatoes, peas, and barley. [3] The method for producing the protein according to [1] or [2], wherein the Fc-binding protein is a polypeptide selected from any of (i) to (ix) below: (i) A polypeptide comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 3 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 4; (ii) A polypeptide having antibody-binding activity, comprising at least the amino acid residues from alanine at position 24 to serine at position 297 of the amino acid sequence described in SEQ ID NO: 3 and the amino acid residues from isoleucine at position 21 to methionine at position 119 of the amino acid sequence described in SEQ ID NO: 4, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions within these amino acid residues; (iii) A polypeptide having antibody-binding activity, comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 3 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 4, provided that it has 70% or more identity with the entire amino acid sequence from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 3 and the entire amino acid sequence from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 4; (iv) Fc-binding proteins comprising amino acid residues from alanine position 24 to serine position 297 in the amino acid sequence described in SEQ ID NO: 3 and amino acid residues from isoleucine position 21 to methionine position 119 in the amino acid sequence described in SEQ ID NO: 4, wherein at least one of the amino acid substitutions shown in (1) to (7) below occurs at said amino acid residues; (1) The 71st cysteine ​​in sequence number 3 is replaced with arginine. (2) The 78th asparagine molecule in SEQ ID NO: 3 is replaced with aspartic acid. (3) Arginine at position 192 of sequence number 3 is replaced with leucine. (4) The 196th asparagine molecule in SEQ ID NO: 3 is replaced with aspartic acid. (5) The glutamine at position 232 of sequence number 3 is replaced with leucine. (6) The 274th cysteine ​​in sequence number 3 is replaced with serine. (7) The lysine at position 295 of sequence number 3 is replaced with glutamic acid. (v) An Fc-binding protein having antibody-binding activity, comprising amino acid residues from alanine at position 24 to serine at position 297 of the amino acid sequence described in Sequence ID No. 3 and amino acid residues from isoleucine at position 21 to methionine at position 119 of the amino acid sequence described in Sequence ID No. 4, wherein the amino acid substitutions described in (1) to (7) above occur at said amino acid residues, and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions described in (1) to (7) above; (vi) An amino acid sequence having 70% or more identity with the entire amino acid sequence in which the amino acid substitutions described in (1) to (7) occur, in the amino acid sequence from the 24th alanine to the 297th serine of the amino acid sequence described in SEQ ID NO: 3 and the amino acid sequence from the 21st isoleucine to the 119th methionine of the amino acid sequence described in SEQ ID NO: 4, and which includes an amino acid sequence in which the amino acid substitutions described in (1) to (7) remain, and which is an Fc-binding protein having antibody-binding activity; (vii) An Fc-binding protein comprising amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 3 and amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 4, wherein at least the amino acid substitutions shown in (1) to (7) and (8) to (10) below occur in said amino acid residues; (8) The valine at position 80 in sequence number 3 is replaced with aspartic acid. (9) The 96th lysine in sequence number 3 is replaced with glutamic acid. (10) The asparagine at position 172 of SEQ ID NO: 3 is replaced with aspartic acid. (viii) From alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that the amino acid substitutions shown in the above (1) to (10) occur in the amino acid residues, and further, in addition to the amino acid substitutions shown in the above (1) to (10), one or several amino acid residues are substituted, deleted, inserted or added at one or several positions, and it is an Fc-binding protein having antibody-binding activity; amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that the amino acid substitutions shown in the above (1) to (10) occur in the amino acid residues, and further, in addition to the amino acid substitutions shown in the above (1) to (10), one or several amino acid residues are substituted, deleted, inserted or added at one or several positions, and it is an Fc-binding protein having antibody-binding activity; substitutions shown in the above (1) to (10) occur in the amino acid residues, and further, in addition to the amino acid substitutions shown in the above (1) to (10), one or several amino acid residues are substituted, deleted, inserted or added at one or several positions, and it is an Fc-binding protein having antibody-binding activity; substitutions shown in the above (1) to (10) occur in the amino acid residues, and further, in addition to the amino acid substitutions shown in the above (1) to (10), one or several amino acid residues are substituted, deleted, inserted or added at one or several positions, and it is an Fc-binding protein having antibody-binding activity; substitutions shown in the above (1) to (10) occur in the amino acid residues, and further, in addition to the amino acid substitutions shown in the above (1) to (10), one or several amino acid residues are substituted, deleted, inserted or added at one or several positions, and it is an Fc-binding protein having antibody-binding activity; substitutions shown in the above (1) to (10) occur in the amino acid residues, and further, in addition to the amino acid substitutions shown in the above (1) to (10), one or several amino acid residues are substituted, deleted, inserted or added at one or several positions, and it is an Fc-binding protein having antibody-binding activity; (ix) An amino acid sequence having 70% or more identity with the entire amino acid sequence in which the amino acid substitutions shown in the above (1) to (10) occur, from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that the amino acid sequence contains the amino acid sequence in which the amino acid substitutions shown in the above (1) to (10) remain, and it is an Fc-binding protein having antibody-binding activity. amino acid sequence from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that the amino acid substitutions shown in the above (1) to (10) occur in the amino acid sequence, and further, in addition to the amino acid substitutions shown in the above (1) to (10), one or several amino acid residues are substituted, deleted, inserted or added at one or several positions, and it is an Fc-binding protein having antibody-binding activity; amino acid sequence from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that the amino acid substitutions shown in the above (1) to (10) occur in the amino acid sequence, and further, in addition to the amino acid substitutions shown in the above (1) to (10), one or several amino acid residues are substituted, deleted, inserted or added at one or several positions, and it is an Fc-binding protein having antibody-binding activity; amino acid sequence from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that the amino acid substitutions shown in the above (1) to (10) occur in the amino acid sequence, and further, in addition to the amino acid substitutions shown in the above (1) to (10), one or several amino acid residues are substituted, deleted, inserted or added at one or several positions, and it is an Fc-binding protein having antibody-binding activity; amino acid sequence from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that the amino acid substitutions shown in the above (1) to (10) occur in the amino acid sequence, and further, in addition to the amino acid substitutions shown in the above (1) to (10), one or several amino acid residues are substituted, deleted, inserted or added at one or several positions, and it is an Fc-binding protein having antibody-binding activity; amino acid sequence from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that the amino acid substitutions shown in the above (1) to (10) occur in the amino acid sequence, and further, in addition to the amino acid substitutions shown in the above (1) to (10), one or several amino acid residues are substituted, deleted, inserted or added at one or several positions, and it is an Fc-binding protein having antibody-binding activity; [4] The production method according to any one of [1] to [3], wherein the medium is TB (Terrific Broth) medium or 2×YT medium. [Advantages of the Invention]

[0012] According to the present disclosure, an efficient production method of an Fc-binding protein can be provided. [Brief Description of the Drawings]

[0013] [Figure 1] A diagram comparing the FcRn production amounts (purified yields) due to differences in strains and media in Example 1 and Comparative Example 1. [Modes for carrying out the invention]

[0014] The present invention will be described in detail below.

[0015] Preferred embodiments of the Fc-binding protein to be produced include polypeptides shown in any of the following (i) to (ix). (i) A polypeptide comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 3 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 4. (ii) A polypeptide having antibody-binding activity, comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 3 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 4, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions within these amino acid residues. (iii) A polypeptide having antibody-binding activity, comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 3 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 4, provided that it has 70% or more identity with the entire amino acid sequence from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 3 and the entire amino acid sequence from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 4. (iv) Fc-binding proteins comprising amino acid residues from alanine position 24 to serine position 297 in the amino acid sequence described in SEQ ID NO: 3 and amino acid residues from isoleucine position 21 to methionine position 119 in the amino acid sequence described in SEQ ID NO: 4, wherein at least one of the amino acid substitutions shown in (1) to (7) below occurs at said amino acid residues; (1) The 71st cysteine ​​in sequence number 3 is replaced with arginine. (2) The 78th asparagine molecule in SEQ ID NO: 3 is replaced with aspartic acid. (3) Arginine at position 192 of sequence number 3 is replaced with leucine. (4) The 196th asparagine molecule in SEQ ID NO: 3 is replaced with aspartic acid. (5) The glutamine at position 232 of sequence number 3 is replaced with leucine. (6) The 274th cysteine ​​in sequence number 3 is replaced with serine. (7) Lysine at position 295 of sequence number 3 is replaced with glutamic acid. (v) An Fc-binding protein having antibody-binding activity, comprising amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 3 and amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 4, wherein the amino acid substitutions described in (1) to (7) above occur at said amino acid residues, and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions described in (1) to (7) above. (vi) An Fc-binding protein having antibody-binding activity, wherein the amino acid sequence from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 3 and the amino acid sequence from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 4 has an amino acid sequence that has 70% or more identity with the entire amino acid sequence in which the amino acid substitutions described in (1) to (7) above occur, and which includes an amino acid sequence in which the amino acid substitutions described in (1) to (7) above remain. (vii) An Fc-binding protein comprising amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 3 and amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 4, wherein at least the amino acid substitutions shown in (1) to (7) and (8) to (10) below occur in said amino acid residues; (8) The valine at position 80 in sequence number 3 is replaced with aspartic acid. (9) The 96th lysine in sequence number 3 is replaced with glutamic acid. (10) The 172nd asparagine molecule in sequence number 3 is replaced with aspartic acid. (viii) From the 24th alanine to the 297th amino acid in the amino acid sequence described in Sequence ID No. 3 The amino acid residues up to serine and the 21st amino acid of the amino acid sequence described in SEQ ID NO: 4 It contains amino acid residues from soleicine to methionine at position 119, provided that the amino acid The amino acid substitutions shown in (1) to (10) above have occurred in the residue, and furthermore, (1 In addition to the amino acid substitutions shown from (10) to (10), one or more amino acid substitutions at one or more positions One or more substitutions, deletions, insertions, and additions of amino acid residues occur, and antibody binding occurs. An active Fc-binding protein. (ix) Of the amino acid sequence described in Sequence ID No. 3, from the 24th alanine to the 297th sec The amino acid sequence up to phosphorus and the 21st isopropyl amino acid in the amino acid sequence described in SEQ ID NO: 4 In the amino acid sequence from isine to the 119th methionine, (1) to (10) The amino acid sequence in which the amino acid substitution shown in ) occurs has more than 70% identity with the entire amino acid sequence. an amino acid sequence in which the amino acid substitutions (1) to (10) described above remain An Fc-binding protein that contains and has antibody-binding activity.

[0016] The amino acid sequence described in Sequence ID No. 3 is the amino acid sequence of the human FcRnα chain (UniProt No. P55899), and the amino acid sequence described in Sequence ID No. 4 is the amino acid sequence of the human FcRnβ chain (UniProt No. P61769). Furthermore, the amino acid residues from alanine (A) at position 24 to serine (S) at position 297 in the amino acid sequence described in Sequence ID No. 3 correspond to the extracellular (EC) region of the human FcRnα chain, and the amino acid residues from isoleucine (I) at position 21 to methionine (M) at position 119 in the amino acid sequence described in Sequence ID No. 4 correspond to the β2 microglobulin (B2M) region of the human FcRnβ chain.

[0017] The polypeptides described in any of (i) to (ix) above only need to include at least the regions corresponding to the EC region of the human FcRnα chain and the B2M region of the human FcRnβ chain described above. For example, they may include all or part of the signal peptide region on the N-terminal side of the EC region of the human FcRnα chain or the B2M region of the human FcRnβ chain, or they may include all or part of the transmembrane region and intracellular region on the C-terminal side of the EC region of the human FcRnα chain.

[0018] In this specification, an Fc-binding protein containing at least amino acid residues corresponding to the EC region of a human FcRnα chain and the B2M region of a human FcRnβ chain is defined as a protein whose amino acid sequence contains at least the amino acid sequence corresponding to the EC region of a human FcRnα chain and the amino acid sequence corresponding to the B2M region of a human FcRnβ chain, and the order of the amino acid residues corresponding to the EC region of a human FcRnα chain and the amino acid residues corresponding to the B2M region of a human FcRnβ chain is irrelevant. That is, the amino acid residues corresponding to the B2M region of a human FcRnβ chain may be located on the N-terminal side or the C-terminal side of the amino acid residues corresponding to the EC region of a human FcRnα chain. Furthermore, the amino acid residues corresponding to the EC region of a human FcRnα chain and the amino acid residues corresponding to the B2M region of a human FcRnβ chain may be directly linked, or they may be linked via a known linker such as a GS linker (a linker consisting of repeating oligopeptides consisting of glycine (G) 4 residues and serine (S) 1 residue).

[0019] In (ii), (v), and (viii) above, "one or several" means one or more amino acids, although this can vary depending on the position of amino acid substitutions and the type of amino acid residues in the three-dimensional structure of the Fc-binding protein. For example, it may mean one to 50, one to 30, one to 20, one to 10, one to 9, one to 8, one to 7, one to 6, one to 5, one to 4, one to 3, one to 2, or one amino acid residue. Modifications of "one or several" amino acid residues may occur in positions other than those disclosed in Japanese Patent Publication No. 2018-183087, Japanese Patent Publication No. 2021-073883, Japanese Patent Publication No. 2021-136967, and Japanese Patent Publication No. 2022-076998, as long as Fc-binding activity is maintained.

[0020] Furthermore, the "modification of one or more amino acid residues" in (ii), (v), and (viii) above may include not only the amino acid modifications at specific positions mentioned above, but also conservative substitutions, which occur between amino acids with similar physical and / or chemical properties. It is generally known to those skilled in the art that conservative substitutions maintain the function of the protein between the substituted and unsubstituted parts. Examples of conservative substitutions include substitutions between glycine and alanine, serine and proline, or glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005). In addition, the "modification of one or more amino acid residues" in (ii), (v), and (viii) above also includes naturally occurring mutations (mutants or variants).

[0021] As an example of (ii), (v), and (viii) above, for example, Fc-binding protein disclosed in Japanese Patent Publication No. 2018-183087, Fc-binding protein disclosed in Japanese Patent Publication No. 2021-073883, Fc-binding protein disclosed in Japanese Patent Publication No. 2021-136967, Examples include the Fc-binding protein disclosed in Japanese Patent Publication No. 2022-076998.

[0022] The amino acid sequence identity in (iii), (vi), and (ix) above only needs to be 70% or more, but it may have a higher degree of identity, for example, 80% or more, 85% or more, 90% or more, or 95% or more.

[0023] In this specification, "identity" of amino acid sequences refers to the percentage obtained by aligning two amino acid sequences to be compared so that as many amino acid residues as possible match, and then dividing the number of matching amino acid residues by the total number of amino acid residues. When aligning, gaps are inserted into one or both of the two sequences to be compared as needed. The method of aligning sequences is not particularly limited, but it can be done using well-known sequence comparison programs such as BLAST (Basic Local Alignment Search Tool), FASTA, or CLUSTAL W. When gaps are inserted, the total number of amino acid residues is the number of residues counted with each gap as one amino acid residue. If the total number of amino acid residues counted in this way differs between the two sequences to be compared, the sequence identity (%) is calculated by dividing the number of matching amino acid residues by the total number of amino acid residues of the longer sequence.

[0024] The Fc-binding protein may have an oligopeptide added to its N-terminus or C-terminus that is useful for accelerating analysis and purification from solutions containing impurities, or for stabilizing the protein. Examples of such oligopeptides include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid, and C-myc tags.

[0025] The N-terminus of the Fc-binding protein may be modified to include a signal peptide to promote efficient expression in the host. Examples of such signal peptides include PelB, DsbA, DsbC, MalE, and TorT, which stimulate protein secretion into the periplasm (Japanese Patent Publication No. 2011-097898). In particular, (I) A polynucleotide encoding the native OmpA signal peptide (regions 1 through 21 of UniProt No. P0A910), or (II) Oligonucleotides encoding polypeptides in which one or several residues of the signal peptide described in (I) above have been modified (one or more substitutions, deletions, insertions, or additions have occurred), By using this method, production can be made more efficiently.

[0026] In this disclosure, an Fc-binding protein is produced by culturing genetically modified Escherichia coli containing a polynucleotide encoding an Fc-binding protein, expressing the protein, and then recovering the expressed protein.

[0027] The present invention is characterized in that the Escherichia coli strain having a gene containing a polynucleotide encoding an Fc-binding protein is strain JM109, and that a specific culture medium is used for culturing said Escherichia coli.

[0028] Examples of JM109 strains include E. coli JM109 Competent Cells (manufactured by Takara Bio Inc.), ECOS Competent E.coli JM109 (manufactured by Nippon Gene Inc.), JM109 Competent Cells (manufactured by Promega Inc.), Champion109 High (manufactured by SMOBIO Inc.), and Competent high JM109 (manufactured by Toyobo Inc.).

[0029] The specific culture medium is one that contains at least peptone and 10 g / L or more of yeast extract. It is more preferable that the specific culture medium contains 15 g / L or more of yeast extract, and even more preferable that it contains 20 g / L or more. Examples of yeast extracts include dried yeast extract (manufactured by Nacalai Tesque), Bacto Yeast Extract (manufactured by Thermo Fisher Scientific), Gibco Yeast Extract (manufactured by Thermo Fisher Scientific), Difco TC Yeastolate UF (manufactured by Thermo Fisher Scientific), Bacto TC Yeastolate (manufactured by Thermo Fisher Scientific), Yeast100 (manufactured by Thermo Fisher Scientific), and Yeast Extract Red Label (manufactured by Oriental Yeast Co., Ltd.).

[0030] The specific culture medium may be, for example, TB (Terrific Broth) medium or 2×YT medium.

[0031] TB medium may contain 23.6 g / L or more of yeast extract, 11.8 g / L or more of tryptone, 9.4 g / L or more of K2HPO4, 2.2 g / L or more of KH2PO4, and 0.4% or more of glycerol. Alternatively, TB medium such as Terrific Broth (manufactured by Thermo Fisher) may be used.

[0032] The 2×YT medium may contain 16 g / L or more of yeast extract, 10 g / L or more of tryptone, and 5 g / L or more of NaCl.

[0033] Peptones include, for example, Tryptone (Nacalai Tesque), Tryptone (Sigma-Aldrich), Phytone Peptone (Thermo Fisher Scientific), Difco Phytone Supplement, UF (TF: Thermo Fisher Scientific), Difco Soytone (TF), Bacto Malt Extract (TF), Wheat 100 UF (TF), Cotton 100 UF (TF), Cotton 200 UF (TF), Soy 100 (TF), Wheat Peptone (Solabia Biotechnology), Broadbean Peptone (Solabia Biotechnology), Lupin Peptone (Solabia Biotechnology), Potato Peptone (Solabia Biotechnology), Pea Peptone (Solabia Biotechnology), Cotton Seed Powder (Himedia), Nutralys F85F (Roquette), and Proyield Soy. Examples include SE50MK (Frieslandcampina), Proyield Soy SE70M-UF (Frieslandcampina), Proyield Wheat WGE80M-UF (Frieslandcampina), Proyield Pea PCE80B (Frieslandcampina), Hiveg Peptone RM001V (Frieslandcampina), Sollis 095E (Roquette), and Hypolypeptone NS (Shiotani MS).

[0034] The aforementioned peptone is more preferable if its origin is one of soybeans, cotton, broad beans, lupin beans, corn, potatoes, peas, or barley, as it does not contain any animal-derived components, thus making it easier to adapt to industrial protein production.

[0035] Examples of soybean-derived peptones include Phytone Supplement, UF (TF Corporation), Phytone Peptone (TF Corporation), Soytone (TF Corporation), Soy 100 (TF Corporation), Proyield Soy SE50MK (FrieslandCampina), Proyield Soy SE70M-UF (FrieslandCampina), and High Polypeptone NS (Shioya MS Co.).

[0036] Examples of cotton-derived peptones include Cotton 100,UF (TF Corporation), Cotton 200,UF (TF Corporation), and Cotton Seed Powder (HiMedia Corporation).

[0037] Examples of broad bean-derived peptones include Broadbean peptone (Solabia Biotechnology) and broad bean peptone (Merck Millipore).

[0038] Examples of peptones derived from lupin beans include Lupin peptone (Solabia).

[0039] Corn-derived peptones include, for example, Solulys 095E (Roquette).

[0040] Potato-derived peptones include, for example, Potato peptone (Solabia).

[0041] Examples of pea-derived peptones include Pea peptone (Solabia), Nutralys F85F (Roquette), Proyield Pea PCE80B (FrieslandCampina), and Vegetable Peptone No. 1 (Oxoid).

[0042] Examples of barley-derived peptone include Malt Extract (TF Corporation).

[0043] Certain culture media may contain peptone at a concentration of 10 g / L or more.

[0044] The method for producing Fc-binding proteins according to this disclosure may include a pre-culture step before culturing using a specific culture medium. Including a pre-culture step is preferable because it makes it easier to control the cell concentration at the start of culture. The culture medium used for culturing E. coli during the pre-culture is not particularly limited, but it is more preferable if it does not contain animal-derived components, as this makes it easier to adapt the protein to industrial production.

[0045] There are no particular limitations on the method of culturing genetically modified Escherichia coli. It may be cultured by batch culture, semi-batch culture (also called fed-batch culture), or perfusion culture, or a combination of these methods. However, if nutrients such as carbon sources and nitrogen sources are added to the culture medium all at once at the start of cultivation, the growth of Escherichia coli and the expression of Fc-binding proteins by the Escherichia coli may be inhibited, and by-products such as organic acids may also be produced, which may adversely affect the efficiency of protein expression and the quality of the obtained protein. Therefore, it is preferable to culture genetically modified Escherichia coli using fed-batch culture, in which the amount of nutrients added at the start of cultivation is kept to a minimum, and nutrients are supplied as needed (fed-batch) during cultivation.

[0046] In this invention, the culture conditions for genetically modified Escherichia coli are not particularly limited as long as the E. coli can grow and express Fc-binding proteins. However, the culture temperature is preferably between 15°C and 50°C, and particularly preferably between 20°C and 33°C. The pH is preferably between 6 and 8. The culture time can be set arbitrarily, but is usually set to several hours or more and 100 hours or less.

[0047] Furthermore, if the genetically modified E. coli contains an inducible promoter and expresses an Fc-binding protein under the control of the promoter, it is preferable to induce the expression of the protein so that it is expressed properly. Examples of inducible promoters that function in E. coli include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, and lpp promoter. For expression induction, for example, an inducer can be used depending on the type of promoter. An example of an inducer is IPTG (Isopropyl-β-D-thiogalactopyranoside). Specifically, when the turbidity of the culture medium (absorbance at 600 nm) is 0.03 or more and 2 or less, an appropriate amount of IPTG can be added, and the expression of the protein of the present invention can be induced by continuing the culture. The concentration of IPTG to be added is, for example, 0.005 mmol / L or more and 1.0 mmol / L or less, preferably 0.01 mmol / L or more and 0.5 mmol / L or less, at the final concentration. Furthermore, when IPTG is added, it is preferable to have an absorbance of 0.03 or more and 2 or less at 600 nm of the culture medium, preferably 0.05 or more and 1 or less, as this allows for good expression of Fc-binding proteins.

[0048] To recover the Fc-binding protein expressed by the method described above, the protein can be isolated and purified from the culture using a method suitable for the protein's expression in genetically modified E. coli. For example, if the protein is expressed in the culture supernatant, the bacterial cells can be separated by centrifugation, and the Fc-binding protein can be purified from the resulting culture supernatant. Alternatively, if the protein is expressed intracellularly (including in the periplasm), the bacterial cells can be collected by centrifugation, then the cells can be disrupted by adding an enzyme treatment agent or surfactant, and the Fc-binding protein can be extracted and then purified.

[0049] To purify the recovered Fc-binding protein, methods known in the art can be used, one example being separation / purification using liquid chromatography. Liquid chromatography includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc., and by combining these chromatography methods, the protein can be prepared in high purity.

[0050] As described above, the amount of Fc-binding protein produced can be calculated by measuring the absorbance of the purified Fc-binding protein solution at a wavelength of 280 nm. [Examples]

[0051] The present invention will be described in detail below using examples and comparative examples, but the present invention is not limited to these examples.

[0052] Example 1: Examination of bacterial strains We evaluated the effect of different E. coli strains on Fc-binding protein production.

[0053] (1) One of the E. coli strains shown in Table 1 was transformed with an expression vector containing a polynucleotide (SEQ ID NO: 2) encoding the Fc-binding protein FcRn_m7GS, which consists of the amino acid sequence described in SEQ ID NO: 1, and an inducible promoter, thereby producing genetically modified E. coli capable of expressing the protein (hereinafter also referred to as FcRn_m7GS-expressing E. coli). FcRn_m7GS (SEQ ID NO: 1) is a polypeptide consisting of the amino acid sequence described in SEQ ID NO: 5, linked from the N-terminus in the following order: β2 microglobulin region of the natural human neonatal Fc receptor (human FcRn) β chain (amino acid residues from positions 21 to 119 of SEQ ID NO: 4 (UniProt No. P61769)) - GS linker (a polypeptide consisting of five linked oligopeptides, each consisting of four glycine (G) residues and one serine (S) residue) - extracellular region of the natural human FcRn α chain (amino acid residues from positions 24 to 297 of SEQ ID NO: 3 (UniProt No. P55899)). The polypeptide undergoes the following amino acid substitutions (i) to (vii), and an OmpA signal peptide (amino acid residues from positions 1 to 21 of UniProt No. P0A910) is added to the N-terminus, and a tag consisting of six histidine (H) residues and a cysteine ​​tag consisting of the amino acid sequence described in SEQ ID NO: 6 are added to the C-terminus. (i) The cysteine ​​(C) at position 172 of sequence number 5 (corresponding to position 193 in sequence number 1 and position 71 in sequence number 3) is replaced with arginine (R). (ii) The asparagine (N) at position 179 of SEQ ID NO: 5 (corresponding to position 200 in SEQ ID NO: 1 and position 78 in SEQ ID NO: 3) is replaced with aspartic acid (D). (iii) The arginine (R) at position 293 of sequence number 5 (corresponding to position 314 in sequence number 1 and position 192 in sequence number 3) is replaced with leucine (L). (iv) The asparagine (N) at position 297 of SEQ ID NO: 5 (corresponding to position 318 in SEQ ID NO: 1 and position 196 in SEQ ID NO: 3) is replaced with aspartic acid (D). (v) The glutamine (Q) at position 333 in sequence number 5 (corresponding to position 354 in sequence number 1 and position 232 in sequence number 3) is replaced with leucine (L). (vi) The cysteine ​​(C) at position 375 of SEQ ID NO: 5 (corresponding to position 396 in SEQ ID NO: 1 and position 274 in SEQ ID NO: 3) is replaced by serine (S). (vii) Lysine (K) at position 396 of SEQ ID NO: 5 (corresponding to position 417 in SEQ ID NO: 1 and position 295 in SEQ ID NO: 3) is replaced with glutamic acid (E).

[0054] [Table 1]

[0055] (2) FcRn_m7GS-expressing Escherichia coli was inoculated into 2×YT medium (Phyton peptone (Thrmo Fisher Scientific): 16 g / L, yeast extract (Nacalai Tesque): 10 g / L, sodium chloride: 5 g / L, kanamycin sulfate: 50 mg / L) and pre-cultured for 16 hours at 30°C and 130 rpm.

[0056] (3) In this culture, a culture medium containing 10 g / L or more of yeast extract was used. As the culture medium, TB (Terrific Broth) medium (Tryptone (Nacalai Tesque): 12 g / L, yeast extract: 24 g / L, glycerol: 10 g / L, dipotassium hydrogen phosphate: 9.4 g / L, potassium dihydrogen phosphate: 2.2 g / L, kanamycin sulfate: 50 mg / L) was used. The pre-culture solution from (2) was added to this culture medium so that the final OD600 nm was 0.02.

[0057] (4) After culturing at 30°C and 130 rpm for 4 hours, IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 0.05 mmol / L, and the culture was further incubated at 25°C and 130 rpm for 24 hours. After the culture was completed, the bacterial cells from 100 mL of culture medium were collected by centrifugation to obtain a pellet.

[0058] (5) To the cell pellet obtained in (4), 14 mL of cell disruption buffer (50 mmol / L 1,3-bis[tris(hydroxymethyl)methylamino]propane, 150 mmol / L sodium chloride, 2.4 mmol / L magnesium sulfate, 3 kU / L Benzonase Nuclease, 60 mg / L lysozyme, 6 g / L Triton X-100 (trade name), pH 10) was added and shaken for 2 hours at 25°C and 150 rpm.

[0059] (6) The solution obtained in (5) was centrifuged at 15,000 rpm for 20 minutes, and the supernatant was filtered and sterilized through a PVDF (polyvinylidene fluoride) membrane with a pore size of 0.22 μm to be collected as a bacterial cell extract.

[0060] (7) The bacterial cell extract obtained in (6) was purified by the Ni-NTA (Nickel-nitrilotriacetic acid) affinity chromatography method shown below. (7-1) Ni-NTA columns were prepared by packing empty chromatography columns with Ni-NTA agarose (Fujifilm Wako Pure Chemical Industries, Ltd., product number: 141-09764). (7-2) The prepared Ni-NTA column was equilibrated by passing five times the volume of Ni-NTA agarose through equilibration buffer A (50 mmol / L 1,3-bis[tris(hydroxymethyl)methylamino]propane, 150 mmol / L sodium chloride, pH 10). (7-3) The bacterial cell extract recovered in (6) was passed through the equilibrated Ni-NTA column to adsorb FcRn_m7GS onto the Ni-NTA agarose. (7-4) After passing the bacterial cell extract through the Ni-NTA column, five times the volume of Ni-NTA agarose equilibration buffer A was passed through to wash away any unadsorbed proteins. (7-5) After washing, four times the volume of Ni-NTA agarose elution buffer A (50 mmol / L 1,3-bis[tris(hydroxymethyl)methylamino]propane, 150 mmol / L sodium chloride, 300 mmol / L imidazole, pH 10) was passed through the Ni-NTA column, and the eluate was collected as the Ni-NTA affinity purified eluate.

[0061] The Ni-NTA affinity purified eluate recovered in (8)(7) was purified by the IgG affinity chromatography method shown below. The Ni-NTA affinity purified eluate recovered in (8-1)(7) was adjusted to pH 6.5 by adding 2 mol / L phosphoric acid. (8-2) An IgG column was prepared by packing an empty chromatography column with IgG Sepharose (Cytiva, product number: 17096901). (8-3) The prepared IgG column was equilibrated by passing five times the volume of IgG Sepharose through equilibration buffer B (50 mmol / L 1,3-bis[tris(hydroxymethyl)methylamino]propane, 150 mmol / L sodium chloride, pH 6.5). (8-4) The Ni-NTA affinity purified eluate, adjusted to pH 6.5 in (8-1), was passed through the equilibrated IgG column to adsorb FcRn_m7GS onto IgG Sepharose. (8-5) After passing the eluent through the IgG column, equilibration buffer B was passed through it in an amount 10 times that of IgG Sepharose to wash away any unadsorbed proteins. (8-6) After washing, the IgG column was passed through six times the volume of elution buffer B (50 mmol / L 1,3-bis[tris(hydroxymethyl)methylamino]propane, 150 mmol / L sodium chloride, pH 8.5) of IgG Sepharose, and the eluate was collected as the IgG affinity purified eluate.

[0062] (9) The absorbance of the IgG affinity purified eluate recovered in (8) was measured at a wavelength of 280 nm, and the FcRn_m7GS production amount (purified yield) was calculated.

[0063] Comparative Example 1 In Example 1(3), the same procedure as in Example 1 was followed, except that FcRn_m7GS-expressing Escherichia coli was cultured using LB-Miller medium (tryptone: 10 g / L, yeast extract: 5 g / L, sodium chloride: 10 g / L) instead of TB medium, and the FcRn_m7GS production (purified yield) was calculated. In other words, in this culture, a medium that did not contain more than 10 g / L of yeast extract was used.

[0064] The results for Example 1 and Comparative Example 1 are summarized in Figure 1. When the E. coli strain JM109 was used and cultured in a medium containing at least 10 g / L of yeast extract, the FcRn production (purified yield) was significantly improved compared to when other E. coli strains (W3110, BL21, HB101, DH5α) were used or when cultured in a medium that did not contain 10 g / L or more of yeast extract.

Claims

1. A method for producing an Fc-binding protein, comprising the steps of culturing genetically modified Escherichia coli containing a polynucleotide encoding an Fc-binding protein and expressing the protein, and recovering the protein, The method for producing the genetically modified Escherichia coli, wherein the genetically modified Escherichia coli is strain JM109 containing a polynucleotide encoding an Fc-binding protein, and the culture medium used for culturing the genetically modified Escherichia coli contains at least peptone and 10 g / L or more of yeast extract.

2. The manufacturing method according to claim 1, wherein the peptone is derived from any of soybeans, cotton, broad beans, lupin beans, corn, potatoes, peas, and barley.

3. The method for producing the product according to claim 1 or 2, wherein the Fc-binding protein is a polypeptide selected from any of (i) to (ix) below; (i) Polypeptides comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 3 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 4; (ii) A polypeptide having antibody-binding activity, comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 3 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 4, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions within these amino acid residues; (iii) A polypeptide having antibody-binding activity, comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 3 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 4, provided that it has 70% or more identity with the entire amino acid sequence from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 3 and the entire amino acid sequence from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 4; (iv) An Fc-binding protein comprising amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 3 and amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 4, wherein at least one of the following amino acid substitutions (1) to (7) occurs in said amino acid residues; (1) The 71st cysteine ​​in sequence number 3 is replaced with arginine. (2) The 78th asparagine molecule in SEQ ID NO: 3 is replaced with aspartic acid. (3) Arginine at position 192 of sequence number 3 is replaced with leucine. (4) The 196th asparagine molecule in Sequence ID No. 3 is replaced with aspartic acid. (5) The glutamine at position 232 of sequence number 3 is replaced with leucine. (6) The 274th cysteine ​​in sequence number 3 is replaced with serine. (7) Lysine at position 295 of Sequence ID No. 3 is replaced with glutamic acid. (v) An Fc-binding protein having antibody-binding activity, comprising amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 3 and amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 4, wherein the amino acid substitutions described in (1) to (7) above occur at said amino acid residues, and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions described in (1) to (7) above; (vi) An amino acid sequence having 70% or more identity with the entire amino acid sequence in which the amino acid substitutions described in (1) to (7) occur, in the amino acid sequence from the 24th alanine to the 297th serine of the amino acid sequence described in SEQ ID NO: 3 and the amino acid sequence from the 21st isoleucine to the 119th methionine of the amino acid sequence described in SEQ ID NO: 4, and which includes an amino acid sequence in which the amino acid substitutions described in (1) to (7) remain, and which has antibody-binding activity; (vii) An Fc-binding protein comprising amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 3 and amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 4, wherein at least the amino acid substitutions shown in (1) to (7) and (8) to (10) below occur in said amino acid residues; (8) The valine at position 80 of SEQ ID NO: 3 is replaced with aspartic acid. (9) Lysine at position 96 of Sequence ID No. 3 is replaced with glutamic acid. (10) The 172nd asparagine molecule in SEQ ID NO: 3 is replaced with aspartic acid. (viiii) From the 24th alanine to the 297th amino acid in the amino acid sequence described in Sequence ID No. 3 The amino acid residues up to serine and the 21st amino acid in the amino acid sequence described in SEQ ID NO: 4 It contains amino acid residues from soroisine to methionine at position 119, provided that the amino acid The amino acid substitutions shown in (1) to (10) above have occurred in the residue, and furthermore, (1 In addition to the amino acid substitutions shown in (10) from (10), one or more amino acids at one or more positions One or more substitutions, deletions, insertions, and additions of amino acid residues occur, and antibody binding occurs. Active Fc-binding proteins; (ix) The amino acid sequence from the 24th alanine to the 297th sec in the sequence number 3 The amino acid sequence up to phosphorus and the 21st isopropyl amino acid in the amino acid sequence described in SEQ ID NO: 4 In the amino acid sequence from isine to the 119th methionine, (1) to (10 The amino acid sequence in which the amino acid substitution shown in ) occurs has 70% or more identity. an amino acid sequence in which the amino acid substitutions (1) to (10) described above remain An Fc-binding protein that contains and has antibody-binding activity.

4. The manufacturing method according to claim 1 or 2, wherein the culture medium is TB (Terrific Broth) medium or 2×YT medium.

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