JP2012130294A - Antibody-binding protein and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a protein which can stably be produced while maintaining the affinity for an antibody possessed by an Fc-binding protein (an extracellular region of a human FcγRI), and to provide a method for producing the protein using a genetic engineering technique.SOLUTION: There are provided a miniaturized Fc-binding protein comprising a first domain and a second domain on the N-terminal side, and deleted of a third domain on the C-terminal side in the three domains composing the Fc-binding protein; a polynucleotide encoding the protein; a vector containing the polynucleotide; and a transformant obtained by transforming a host with the vector.

Description

  The present invention relates to a protein having a high binding affinity for a human antibody and a method for producing the protein using a genetic engineering technique.

  Antibodies are widely used in medicines, research reagents and the like, and are recognized as essential substances particularly in the medical field. As the use of antibodies expands, the demand for substances that recognize and bind to antibodies is also increasing. Among them, a human Fc receptor (hereinafter simply abbreviated as “FcR”) has a high binding affinity for human antibody IgG, and therefore has been industrially applied (Patent Document 1).

  FcR has subclasses of FcγRI, FcγRIIa, FcγRIIb, and FcγRIII from the functional and structural classification after complex formation with an antibody. These FcRs are proteins that play a central role in the immune system, such as being present on the surface of immune cells and binding to antibodies to form a complex, whereby signals are transmitted into immune cells. The homology of the amino acid sequence between FcR subclasses is very high, and it is considered that the amino acid position that actually interacts with an antibody can be estimated in the formation of a complex with the antibody. On the other hand, in the extracellular region of FcγRI, which is one of the FcR subclasses (hereinafter referred to as “Fc binding protein”), a third domain that does not exist in other FcRs is present on the C-terminal side. (Non-Patent Documents 1 and 2), it is considered that FcγRI has high affinity and antibody selectivity due to the presence of this third domain.

Japanese translation of PCT publication No. 2002-531086 JP 2008-245580 A

J. et al. M.M. Allen et al., Science, 243, 378, 1989 A. Paetz et al., Biochem. Biophys. Res. Commun. , 338, 1811, 2005

  When an Fc-binding protein is produced and used for the purpose of using it as an antibody detection reagent or the like, it has been expressed (manufactured) and used as a protein containing all three domains of the Fc-binding protein (Patent Document 2). However, the expression level of the Fc binding protein is low, and there are problems with respect to stability and the like.

  Therefore, the present invention is to provide a protein that can be stably produced while maintaining the affinity for the antibody of the Fc-binding protein, and a method for producing the protein using a genetic engineering technique.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have shown that FcγRI has high affinity and antibody selectivity among the three domains constituting the extracellular region (Fc binding protein) of FcγRI. Even if the third domain on the C-terminal side, which is considered to be a cause, is deleted (that is, only the first domain and the second domain on the N-terminal side), it has high affinity for the antibody. As a result, the present invention has been completed.

That is, the present invention includes the following aspects:
(1) An Fc-binding protein comprising at least amino acids from the 7th alanine to the 169th threonine in the amino acid sequence shown in SEQ ID NO: 52.

  (2) Fc binding comprising an amino acid sequence of at least the 7th alanine to the 169th threonine in the amino acid sequence shown in SEQ ID NO: 52, wherein one or more of the amino acids are substituted with other amino acids Sex protein.

  (3) A polynucleotide encoding the amino acid sequence according to (1) or (2).

  (4) A polynucleotide comprising the first to 522th nucleotides in the nucleotide sequence set forth in SEQ ID NO: 7.

  (5) A vector for expressing an Fc-binding protein, comprising the polynucleotide according to (3) or (4).

  (6) A transformant obtained by transforming a host using the vector according to (5).

  (7) The transformant according to (6), wherein the host is Escherichia coli.

  (8) A method for producing an Fc-binding protein using the transformant according to (6) or (7).

  Hereinafter, the present invention will be described in detail.

  As shown in FIG. 1, human FcγRI is composed of a signal peptide region consisting of 15 amino acids from the N-terminal side (SS, region 1 to 15 of the amino acid sequence described in SEQ ID NO: 6), and 277 amino acids. Extracellular region (EC, the region from the 16th to 292th of the amino acid sequence described in SEQ ID NO: 6), transmembrane region consisting of 21 amino acids (TM, 293th to 313 of the amino acid sequence described in SEQ ID NO: 6) Region), an intracellular region consisting of 61 amino acids (C, region 314 to 374 of the amino acid sequence shown in SEQ ID NO: 6). Further, the extracellular region (Fc binding protein) of human FcγRI is the first domain (22 of the amino acid sequence described in SEQ ID NO: 6) from the N-terminal side from the public database (UniProtKB / Swiss-Prot No. P12314). To the 101st region), the second domain (the 95th to 184th region of the amino acid sequence described in SEQ ID NO: 6), the third domain (from the 190th amino acid sequence described in SEQ ID NO: 6) It is known to have (up to 277th area).

The Fc binding protein of the present invention is a SEQ ID NO: which is a partial region of an Fc binding protein (extracellular region of human FcγRI) (a region from the 16th to the 292th of the amino acid sequence described in SEQ ID NO: 6). 52 (the region from the 16th to the 189th of the amino acid sequence described in SEQ ID NO: 6) at least the amino acid from the 7th alanine to the 169th threonine (ie, the first domain and the second domain). Domain region). A protein in which one or more of the amino acids constituting the Fc-binding protein of the present invention is substituted with another amino acid is also included in the Fc-binding protein of the present invention. One aspect of the protein is a protein comprising at least the 7th alanine to the 169th threonine of the amino acid sequence set forth in SEQ ID NO: 52,
(A) The 10th threonine of SEQ ID NO: 52 is replaced with lysine (a) The 23rd threonine of SEQ ID NO: 52 is replaced with alanine or serine (c) The 31st leucine of SEQ ID NO: 52 is replaced with arginine or proline ( D) 47th alanine of SEQ ID NO: 52 is replaced with valine (e) 48th threonine of SEQ ID NO: 52 is replaced with isoleucine (f) 54th serine of SEQ ID NO: 52 is replaced with phenylalanine or threonine (g) The 56th arginine of No. 52 is substituted with histidine (g) The 62nd valine of SEQ ID No. 52 is substituted with alanine or glutamic acid (g) The 63rd asparagine of SEQ ID No. 52 is substituted with aspartic acid (co) SEQ ID No. 52 The aspartic acid 79 in the above was replaced with glutamic acid. Soleucine was replaced with valine (S) 95th serine of SEQ ID NO: 52 was replaced with asparagine (S) 99th phenylalanine of SEQ ID NO: 52 was replaced with leucine (S) 110th histidine of SEQ ID NO: 52 was replaced with arginine (So) 116th leucine of SEQ ID NO: 52 is replaced with arginine or proline (t) 134th tryptophan of SEQ ID NO: 52 is replaced with leucine (h) 141st leucine of SEQ ID NO: 52 is replaced with proline (tu) The 145th isoleucine of SEQ ID NO: 52 is substituted with methionine (te) The protein includes one or more amino acid substitutions of 148th asparagine of SEQ ID NO: 52 substituted with serine (Japanese Patent Application No. 2010-052789) ). Further, even if a tag peptide for purification such as polyhistidine tag is added to the N-terminal side or C-terminal side of the Fc-binding protein of the present invention, it is included in the Fc-binding protein of the present invention.

As a method for producing a polynucleotide encoding the Fc-binding protein of the present invention (hereinafter simply referred to as “polynucleotide of the present invention”),
(I) a method of converting the amino acid sequence of the Fc-binding protein of the present invention into a nucleotide sequence and artificially synthesizing a polynucleotide containing the nucleotide sequence;
(II) A polynucleotide containing the whole or a partial sequence of human FcγRI is prepared directly or artificially from human FcγRI cDNA using a DNA amplification method such as PCR, and the prepared polynucleotides are ligated by an appropriate method. And how to make
Can be illustrated. In addition, when converting from an amino acid sequence to 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). However, since CCC is less frequently used in proline (Pro) (because it is a so-called rare codon), it may be converted so as to avoid those codons. Analysis of codon usage frequency can also be performed by using a public database (for example, Codon Usage Database on the website of Kazusa DNA Research Institute). As an example of conversion from the amino acid sequence of the Fc binding protein of the present invention to a nucleotide sequence based on the codon usage of E. coli, among the nucleotide sequence shown in SEQ ID NO: 7 converted from the amino acid sequence shown in SEQ ID NO: 52 Examples thereof include a polynucleotide comprising the first to 522th nucleotides.

  In addition, a polynucleotide encoding a signal peptide may be added to the 5 ′ end side of the polynucleotide of the present invention produced by the method of (I) or (II). When the host is E. coli, the signal peptide PelB (uniprot No. P0C1C1 1st to 22nd area), DsbA (UniProt No. P0AEG4 1st to 19th area), MalE (UniProt No. P0AEX9 1st to 26th area, Examples thereof include signal peptides that secrete proteins into the periplasm, such as SEQ ID NO: 1) and TorT (regions 1 to 18 of UniProt No. P38683) (Japanese Patent Application No. 2009-256180).

  When transforming a host using the polynucleotide of the present invention, the polynucleotide of the present invention itself may be used, but an expression vector (for example, a bacteriophage or cosmid usually used for transformation of prokaryotic cells or eukaryotic cells). It is more preferable to use a product in which the polynucleotide of the present invention is inserted at an appropriate position in a plasmid or a plasmid. The expression vector is not particularly limited as long as it is stably present in the host to be transformed and can be replicated. When Escherichia coli is used as a host, pET plasmid, pUC plasmid, pTrc99a plasmid, pCDF plasmid, pBBR plasmid Etc. can be illustrated. When inserting the polynucleotide of the present invention into the expression vector, a plurality of the polynucleotides of the present invention may be inserted (may include a polynucleotide encoding a linker peptide in the linking portion). Insertion in a linked state can be expected to improve the productivity of the Fc-binding protein of the present invention by the transformant (Japanese Patent Application Nos. 2009-274352 and 2009-274353).

  In order to transform a host using an expression vector into which the polynucleotide of the present invention has been inserted, a method commonly used by those skilled in the art may be used. For example, when a microorganism belonging to the genus Escherichia such as Escherichia coli is selected as a host, it may be transformed by a method such as a heat shock method or an electroporation method. A transformant expressing the Fc-binding protein of the present invention can be obtained by screening the transformant obtained by the above method by an appropriate method. The transformant can be screened, for example, by evaluating the binding activity of the Fc binding protein to the antibody. The binding activity can be evaluated, for example, by measuring the binding activity to IgG using an ELISA method or a surface plasmon resonance method. The IgG used for the measurement is preferably human IgG, more preferably human IgG1.

  The Fc-binding protein of the present invention can be produced by culturing a host (transformant) transformed with an expression vector containing a polynucleotide encoding the protein (polynucleotide of the present invention). The transformant used in the method for producing an Fc-binding protein of the present invention may be cultured in a medium suitable for culturing the target host. When the host is Escherichia coli, LB (Luria-Bertani supplemented with necessary nutrients) is used. ) A medium is an example of a preferable medium. In order to stably culture a transformant holding an expression vector, it is preferable to add a drug corresponding to the drug resistance gene inserted into the expression vector and culture it. When the host is Escherichia coli, the culture temperature is 10 ° C. to 40 ° C., preferably 25 ° C. to 37 ° C., more preferably around 30 ° C., but may be selected depending on the characteristics of the Fc-binding protein to be expressed. When the host is Escherichia coli, the pH of the medium is pH 6.8 to pH 7.4, preferably around pH 7.0.

  When the expression vector containing the polynucleotide of the present invention contains an inducible promoter, it may be induced under conditions that allow the polypeptide containing the Fc-binding protein of the present invention to be expressed well. Examples of the inducer include IPTG (isopropyl-β-D-thiogalactopyranoside). When the host is Escherichia coli, after measuring the turbidity (absorbance at 600 nm) of the culture solution, when the value reaches about 0.5, an appropriate amount of IPTG is added, followed by culturing, whereby the Fc of the present invention Binding protein expression can be induced. The addition concentration of IPTG is preferably a final concentration of 0.005 to 1.0 mM, more preferably a final concentration 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 extract the Fc-binding protein of the present invention from the culture medium of the transformant of the present invention, an extraction method may be selected as appropriate depending on the form of expression. The Fc-binding protein may be extracted from the culture supernatant obtained by separation. On the other hand, when it is expressed intracellularly (including periplasm in prokaryotes), the cells are collected by centrifugation, and then the cells are disrupted by adding ultrasound, an enzyme treatment agent or a surfactant. Then, the Fc binding protein may be extracted.

  In order to separate and purify the Fc-binding protein of the present invention from the extract containing the Fc-binding protein of the present invention, a method known in the art may be used. An example is separation / purification using liquid chromatography. Examples of liquid chromatography include ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography and the like. By performing a purification operation by combining these chromatographies, the Fc-binding protein of the present invention can be prepared with high purity.

The Fc-binding protein of the present invention is a protein comprising at least a first domain and a second domain on the N-terminal side among the three domains possessed by the extracellular region (Fc binding protein) of human FcγRI. In terms of
(1) Among the amino acid sequences set forth in SEQ ID NO: 52, an Fc-binding protein comprising at least amino acids from the 7th alanine to the 169th threonine, or (2) among the amino acid sequences set forth in SEQ ID NO: 52, An Fc-binding protein comprising at least an amino acid from the seventh alanine to the 169th threonine, wherein one or more of the amino acids are replaced with another amino acid,
It is. The Fc-binding protein of the present invention lacks the third domain on the C-terminal side of the Fc-binding protein, which has been considered to be a cause of FcγRI having high affinity and antibody selectivity. However, it retains the high binding affinity for the antibody possessed by the Fc-binding protein, and succeeded in miniaturizing the protein molecule. Therefore, when an Fc-binding protein is produced using a genetic engineering technique, a stable protein can be produced, and the availability of the produced Fc-binding protein for an antibody detection reagent or the like can be increased.

It is a figure which shows the structure of human FcγRI. It is a figure which shows the preparation method and structure of plasmid pETmalE. It is a figure which shows the preparation method and structure of plasmid pETFcR. It is a figure which shows the preparation method and structure of plasmid pETsFcR. It is the figure which showed the binding selectivity to each subclass of human IgG of wild type Fc binding protein and miniaturized Fc binding protein (Fc binding protein of this invention). It is the figure which confirmed the molecular weight of wild type Fc binding protein and miniaturized Fc binding protein (Fc binding protein of this invention). It is the figure which evaluated the affinity to human IgG of miniaturized Fc binding protein (Fc binding protein of this invention).

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.

Example 1
A plasmid into which a polynucleotide encoding a signal peptide was inserted was prepared by the following method.
(1) In order to produce a polynucleotide encoding the MalE signal peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 (MKIKTGARIALLSALTTMMFSASALA), SEQ ID NO: 2 (5′-TATA [CATATAG] AAAAATAAAAACAGGTGCACGCATCC-3 ′; within square brackets The base of is an oligonucleotide consisting of the sequence described in the restriction enzyme NdeI site), an oligonucleotide consisting of the sequence described in SEQ ID NO: 3 (5′-GCATTAACGACGATGAGTTTTCCCGCTCCGGCTCTCCGCC-3 ′), and SEQ ID NO: 4 (5′-ATCGTCGTTAATGCGGGATCTGCGTGGCTGGCTGCGTCGC And an oligonucleotide consisting of the sequence described in SEQ ID NO: 5 (5′-TTGT C [CCATGG] CTTCTCTCATTTTGGCGGAGAGCCG-3 ′; the bases in square brackets are the restriction enzyme NcoI site), and oligonucleotides having the sequences described above were linked by the PCR method. For the PCR reaction, the reaction solution shown in Table 1 was used, and 5 cycles of a reaction comprising 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 as one cycle I did it.

なお、表１に記載のオリゴヌクレオチドミックスは、５０ｐｍｏＬ／μＬの前記４種類（配列番号２から５に記載の配列からなるオリゴヌクレオチド）の合成オリゴヌクレオチド溶液を、同量ずつ採取し混合した溶液を指す。
（２）（１）のＰＣＲ産物を鋳型とし、ＰＣＲプライマーとして配列番号２および配列番号５に記載の配列からなる各オリゴヌクレオチドを用いた他は、表１に記載の反応液と同じ組成を用い、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行なうＰＣＲ反応により、ＭａｌＥシグナルペプチドをコードするポリヌクレオチドを作製した。
（３）（２）で作製したＭａｌＥシグナルペプチドをコードするポリヌクレオチドを、制限酵素ＮｄｅＩとＮｃｏＩで消化した。前記消化物を、あらかじめ制限酵素ＮｄｅＩとＮｃｏＩで消化したｐＥＴ２６ｂ（＋）プラスミドベクター（Ｎｏｖａｇｅｎ社製）にライゲーションし、ヒートショック法により大腸菌（Ｅ．ｃｏｌｉ） ＪＭ１０９株（タカラバイオ社製）を形質転換した。
（４）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを含むＬＢ培地で培養し、形質転換体よりプラスミドＤＮＡを抽出することで、プラスミドｐＥＴＭａｌＥを調製した。ｐＥＴＭａｌＥ作製方法の概略およびｐＥＴＭａｌＥの構造を図２に示す。

The oligonucleotide mix shown in Table 1 is a solution obtained by collecting and mixing the same amount of the above-mentioned four types of synthetic oligonucleotide solutions of 50 pmoL / μL (the oligonucleotides consisting of the sequences described in SEQ ID NOs: 2 to 5). Point to.
(2) The same composition as the reaction solution shown in Table 1 was used except that the PCR product of (1) was used as a template and each oligonucleotide consisting of the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 5 was used as a PCR primer. The MalE signal peptide is encoded by a PCR reaction in which 30 cycles of 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 are performed for 30 cycles A polynucleotide was made.
(3) The polynucleotide encoding the MalE signal peptide prepared in (2) was digested with restriction enzymes NdeI and NcoI. The digested product was ligated to pET26b (+) plasmid vector (manufactured by Novagen) previously digested with restriction enzymes NdeI and NcoI, and Escherichia coli (E. coli) JM109 strain (manufactured by Takara Bio Inc.) was transformed by the heat shock method. did.
(4) The obtained transformant was cultured in an LB medium containing 50 μg / mL kanamycin, and plasmid DNA was extracted from the transformant to prepare plasmid pETmalE. FIG. 2 shows an outline of the method for preparing pETmalE and the structure of pETmalE.

Example 2
A polynucleotide encoding an Fc binding protein to be inserted into the plasmid prepared in Example 1 was prepared.
(1) As a wild-type Fc-binding protein, a protein consisting of amino acids from the 16th glutamine to the 289th valine is selected from the amino acid sequence of the human Fc receptor FcγRI described in SEQ ID NO: 6, The encoding polynucleotide was designed using E. coli codons. The designed polynucleotide sequence is shown in SEQ ID NO: 7.
(2) A synthetic oligonucleotide for producing a polynucleotide having the sequence described in SEQ ID NO: 7 was prepared. The sequences of the prepared oligonucleotides are shown in SEQ ID NOs: 8 to 45.
(3) A polynucleotide encoding a wild-type Fc-binding protein was prepared from the synthetic oligonucleotide prepared in (2) by performing the following two-step PCR.
(3-1) The first stage PCR reaction uses the reaction solution shown in Table 2, and after heat treatment at 94 ° C. for 5 minutes, the first step at 94 ° C. for 30 seconds, the second step at 62 ° C. for 30 seconds, The reaction was carried out by 25 cycles of the third step of 1 minute at 72 ° C. for one cycle, and finally heat-treated at 72 ° C. for 7 minutes.

なお、表２に記載のオリゴヌクレオチドミックスは、５０ｐｍｏＬ／μＬの３８種類（配列番号８から４５に記載の配列からなるオリゴヌクレオチド）の合成オリゴヌクレオチド溶液を同量ずつ採取し混合した溶液を指す。
（３−２）２段階目のＰＣＲ反応は、表３に示す反応液を用い、９４℃で５分間熱処理後、９４℃で３０秒間の第１ステップ、６５℃で３０秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を２５サイクル行ない、最後に７２℃で７分間熱処理することで行なった。

The oligonucleotide mix described in Table 2 refers to a solution obtained by collecting and mixing the same amount of 38 types of synthetic oligonucleotide solutions (oligonucleotides having the sequences described in SEQ ID NOs: 8 to 45) of 50 pmoL / μL.
(3-2) The second stage PCR reaction uses the reaction solution shown in Table 3, and after heat treatment at 94 ° C. for 5 minutes, the first step at 94 ° C. for 30 seconds, the second step at 65 ° C. for 30 seconds, The reaction was carried out by 25 cycles of the third step of 1 minute at 72 ° C. for one cycle, and finally heat-treated at 72 ° C. for 7 minutes.

なお、表３に示す組成のうち、鋳型は１段階目のＰＣＲ産物を用い、ＰＣＲプライマーは配列番号４６（５’−ＴＣＡＧ［ＣＣＡＴＧＧ］ＧＡＣＡＡＧＴＡＧＡＴＡＣＣＡＣＣＡＡＡＧＣＴＧＴＧＡＴＴＡ−３’；角かっこ内は制限酵素ＮｃｏＩサイト）に記載の配列からなるオリゴヌクレオチドおよび配列番号４７（５’−ＣＣ［ＡＡＧＣＴＴ］ＡＡＴＧＡＴＧＡＴＧＡＴＧＡＴＧＡＴＧＧＡＣＣＧＧＧＧＴＣＧＧＣＡＧＴＴＧＡＡＧＡＣＣＣＡＧ−３’；角かっこ内は制限酵素ＨｉｎｄＩＩＩサイト）に記載の配列からなるオリゴヌクレオチドを用いた。なお、配列番号４７に記載の配列からなるＰＣＲプライマーは、野生型Ｆｃ結合性タンパク質のＣ末端側に検出用のタグである６個のヒスチジン（Ｈｉｓ）が付加するよう設計している。
（４）（３−２）でのＰＣＲ産物をアガロースゲルを用いて抽出精製（ＱＩＡｑｕｉｃｋ Ｇｅｌ ｅｘｔｒａｃｔｉｏｎ ｋｉｔ：キアゲン社製）し、野生型Ｆｃ結合性タンパク質をコードするポリヌクレオチドを調製した。

Of the compositions shown in Table 3, the template used was the first-stage PCR product, and the PCR primer was SEQ ID NO: 46 (5′-TCAG [CCATGG] GACAAGTAGATACACCACCAAGCTGTGTATA-3 ′; the restriction enzyme NcoI site in square brackets) The oligonucleotide consisting of the sequence described and the oligonucleotide consisting of the sequence described in SEQ ID NO: 47 (5′-CC [AAGCTT] AATGATGATGATGATGATGGGACCGGGGTCCGCAGGTTGAAGACCCCAG-3 ′; the restriction enzyme HindIII site in square brackets) were used. The PCR primer consisting of the sequence shown in SEQ ID NO: 47 is designed so that six histidines (His) as detection tags are added to the C-terminal side of the wild-type Fc-binding protein.
(4) The PCR product in (3-2) was extracted and purified (QIAquick Gel extraction kit: Qiagen) using an agarose gel to prepare a polynucleotide encoding a wild-type Fc-binding protein.

Example 3
An Fc-binding protein expression plasmid was prepared from the plasmid prepared in Example 1 and the polynucleotide encoding the Fc-binding protein prepared in Example 2.
(1) The pETMalE vector prepared in Example 1 and the polynucleotide encoding the wild-type Fc-binding protein prepared in Example 2 were digested with restriction enzymes NcoI and HindIII, respectively, ligated, and E. coli by the heat shock method. (E. coli) JM109 strain (manufactured by Takara Bio Inc.) was transformed.
(2) After culturing the obtained transformant in an LB medium containing 50 μg / mL kanamycin, a plasmid was extracted according to a conventional method (Qiaprep Spin Miniprep Kit: manufactured by Qiagen), and this was transformed into pETFcR (inserted polynucleotide). The protein encoded by is designated as “wild-type Fc binding protein”). The outline of pETFcR preparation and the structure of pETFcR are shown in FIG.
(3) Escherichia coli (E. coli) BL21 (DE3) strain was transformed with pETFcR by the heat shock method.

Example 4
The sequence of the polynucleotide encoding the Fc-binding protein inserted into pETFcR (FIG. 3) was used for the cycle sequence reaction using the Big Dye Terminator Cycle Sequencing FS read Reaction kit (PE Applied Biosystems) based on the chain terminator method. And analyzed with a fully automatic DNA sequencer ABI Prism 3700 DNA analyzer (manufactured by PE Applied Biosystems). As a sequencing primer, an oligonucleotide consisting of the sequence shown in SEQ ID NO: 48 (5′-TAATACGACTCACTATAGGGG-3 ′) and an oligonucleotide consisting of the sequence shown in SEQ ID NO: 49 (5′-TATGCTAGTTATTGCTCAGAG-3 ′) are used. It was. As a result of the analysis, it was confirmed that the sequence of the polynucleotide encoding the Fc binding protein inserted into pETFcR was as designed.

Example 5
An Fc-binding protein expression plasmid was prepared from the plasmid prepared in Example 1 and a polynucleotide encoding the Fc-binding protein of the present invention.
(1) Using the plasmid pETFcR prepared in Example 3 as a template, using the reaction solution shown in Table 4, the PCR reaction was heat-treated at 98 ° C. for 5 minutes, then the first step at 98 ° C. for 10 seconds, and 5 ° C. at 55 ° C. The reaction was performed by performing 30 cycles of the second step for 2 seconds and the third step for 1 minute at 72 ° C. for 1 cycle, and finally performing heat treatment at 72 ° C. for 5 minutes.

なお、表４に示す組成のうち、ＰＣＲプライマーは配列番号４６に記載の配列からなるオリゴヌクレオチドおよび配列番号５０（５’−ＣＣＧ［ＣＴＣＧＡＧ］ＧＣＴＧＣＣＧＣＣＡＡＡＣＡＧＴＴＣＴＴＴＧＡＣＧＧ−３’；角かっこ内は制限酵素ＸｈｏＩサイト）に記載の配列からなるオリゴヌクレオチドを用いた。
（２）（１）のＰＣＲ産物をアガロースゲルを用いて抽出精製（ＱＩＡｑｕｉｃｋ Ｇｅｌ ｅｘｔｒａｃｔｉｏｎ ｋｉｔ：キアゲン社製）し、ポリヌクレオチドを調製した。
（３）精製したポリヌクレオチドと、実施例１に調製したプラスミドｐＥＴＭａｌＥとを制限酵素ＮｃｏＩおよびＸｈｏＩで消化し、ライゲーション後、ヒートショック法により大腸菌（Ｅ．ｃｏｌｉ） ＪＭ１０９株（タカラバイオ社製）を形質転換した。
（４）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを含むＬＢ培地にて培養後、定法に従いプラスミドを抽出し（Ｑｉａｐｒｅｐ Ｓｐｉｎ Ｍｉｎｉｐｒｅｐ Ｋｉｔ：キアゲン社製）、これをｐＥＴｓＦｃＲと命名した。プラスミドｐＥＴｓＦｃＲ作製の概略とｐＥＴｓＦｃＲの構造を図４に示す。

In the composition shown in Table 4, the PCR primer is an oligonucleotide having the sequence set forth in SEQ ID NO: 46 and SEQ ID NO: 50 (5′-CCG [CTCGAG] GCTGCCGCCAAACAGATTCTTTGACGG-3 ′; the restriction enzyme XhoI site in square brackets) An oligonucleotide having the sequence described in 1) was used.
(2) The PCR product of (1) was extracted and purified (QIAquick Gel extraction kit: Qiagen) using an agarose gel to prepare a polynucleotide.
(3) The purified polynucleotide and the plasmid pETMalE prepared in Example 1 were digested with restriction enzymes NcoI and XhoI, ligated, and then E. coli JM109 strain (manufactured by Takara Bio Inc.) was obtained by the heat shock method. Transformed.
(4) The obtained transformant was cultured in an LB medium containing 50 μg / mL kanamycin, and then a plasmid was extracted according to a conventional method (Qiaprep Spin Miniprep Kit: manufactured by Qiagen), which was named pETsFcR. FIG. 4 shows an outline of the construction of plasmid pETsFcR and the structure of pETsFcR.

  When the sequence of pETsFcR was confirmed by the method described in Example 4, it was confirmed that the sequence of the polynucleotide encoding the inserted Fc binding protein was as designed. Among the pETsFcRs, a protein containing the MalE signal peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and the Fc binding protein of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 52 (hereinafter referred to as “miniaturized Fc binding protein”) The sequence of the polynucleotide encoding) is shown in SEQ ID NO: 51. Details of SEQ ID NO: 51 are the sequences in which the first to 78th bases encode a MalE signal peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the 79th to 99th bases are linker sequences, and the 100th to 621th positions A sequence encoding the Fc-binding protein of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 52, and a base sequence from the 622nd to the 630th base is a linker sequence. It should be noted that six histidine (His) tags derived from pETmalE are used as detection tags. The pETsFcR whose sequence was confirmed was transformed into E. coli BL21 (DE3) strain by the heat shock method.

Example 6
The plasmid pETFcR transformant prepared in Example 3 and the plasmid pETsFcR transformant prepared in Example 5 were used to express Fc binding protein.
(1) E. coli BL21 (DE3) strain transformed with pETFcR (FIG. 3) or pETsFcR (FIG. 4) was cultured in LB medium containing 50 μg / mL kanamycin (at 18 ° C. at 18 ° C.). After 8 hours, 8 mL of the culture solution was inoculated into LB medium (800 mL) supplemented with 50 μg / mL kanamycin.
(2) When the value of the turbidity (absorbance at 600 nm) of the culture solution changes from 1.0 to 2.0, the culture temperature is switched to 15 ° C., and the IPTG is added to the culture solution so that the final concentration is 0.1 mM. After the addition, the cells were further cultured for 20 hours.
(3) The cells are collected from the culture solution by centrifugation, suspended in 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, and then the protein is removed from the cells by ultrasonic disruption. Extracted, and the supernatant was collected by centrifugation.
(4) A nickel chelate column (His · Bind) equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl in advance, with imidazole added to the collected supernatant to a final concentration of 20 to 50 mM. (Resin: manufactured by Novagen), washed with 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, and 20 mM Tris-HCl buffer (pH 8) containing 500 mM imidazole and 10% glycerin. 0.0) was used to elute the protein.

Example 7
The binding activity of wild-type Fc-binding protein or miniaturized Fc-binding protein purified by the method described in Example 6 to each subclass of IgG (IgG1, IgG2, IgG3, IgG4) is described in the ELISA described below. It was evaluated by the law.
(1) IgG1, IgG2, IgG3, and IgG4 (manufactured by Sigma-Aldrich), which are subclasses of human IgG, were fixed to a well of a 96-well microplate at a concentration of 1 μg / well (4 ° C., 18 hours).
(2) After immobilization, blocking was performed with 50 mM Tris-HCl buffer (pH 8.0) containing 2% skim milk.
(3) After washing with a washing buffer (10 mM Tris-HCl buffer (pH 8.0) containing 0.2% (w / v) Tween 20 and 150 mM NaCl), the prepared protein solution was treated with 50 mM Tris. -Diluted appropriately with HCl buffer (pH 8.0) and reacted with immobilized gamma globulin (1 hour at 30 ° C).
(4) After the reaction was completed, the plate was washed again with the washing buffer, and Horse radish Peroxidase (HRP) -labeled anti-His-Tag antibody reagent (BETHYL) was diluted 10,000 times and added at 100 μL / well.
(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, the color was developed, the reaction was stopped with 1M phosphoric acid solution, and the absorbance at 450 nm was measured.

  The measurement results are shown in FIG. In the figure, the X-axis (horizontal axis) represents the IgG subclass, and the Y-axis (vertical axis) is the absorbance at 450 nm (unit is arbitrary), indicating antibody binding activity. As shown in FIG. 5, the reactivity to each IgG subclass showed reactivity of IgG3> IgG1> IgG4 >> IgG2 for both the wild type Fc binding protein and the miniaturized Fc binding protein. Therefore, it can be said that the miniaturized Fc binding protein which is the Fc binding protein of the present invention has the same performance as the wild type Fc binding protein with respect to binding to IgG.

Example 8
In order to confirm the molecular weight of the miniaturized Fc binding protein, Western blotting shown below was performed.
(1) Wild-type Fc-binding protein or miniaturized Fc-binding protein purified by the method described in Example 6 was subjected to reduction treatment and separated by SDS-PAGE.
(2) After SDS-PAGE, it was transferred to a PVDF membrane (manufactured by GE Healthcare), and the transferred PVDF membrane was blocked with 50 mM Tris-HCl buffer (pH 8.0) containing 5% skim milk.
(3) After washing with a washing buffer (10 mM Tris-HCl buffer (pH 8.0) containing 0.2% (w / v) Tween 20 and 150 mM NaCl), the Horse radish Peroxidase (HRP) labeling Anti-His-Tag antibody reagent (BETHYL) was reacted (1 hour at room temperature).
(4) After completion of the reaction, washing was performed again with the washing buffer, and detection was performed using a detection reagent (ECL Plus: manufactured by GE Healthcare).

  The measurement results are shown in FIG. In the figure, M is a marker, AP is a sample applied to the column in Example 6, FT is a fraction passing through the column, and Fr1 to Fr4 are fractions eluted from the column. The molecular weight calculated from the amino acid composition is about 32 kDa for the wild-type Fc-binding protein and about 21 kDa for the miniaturized Fc-binding protein. From the results shown in FIG. 6, the position of the wild-type Fc-binding protein is about 30 kDa. A band was confirmed at a position of about 20 kDa of the activated Fc binding protein. Therefore, it was found that the miniaturized Fc binding protein, which is the Fc binding protein of the present invention, certainly has a smaller molecular weight than the wild type Fc binding protein.

Example 9
The binding affinity of the miniaturized Fc binding protein to human IgG1 was measured using a surface plasmon resonance method using BIAcoreT100 (manufactured by GE Healthcare).
(1) The miniaturized Fc-binding protein purified by the method described in Example 6 was dialyzed against 10 mM HEPES buffer (pH 7.4) containing 150 mM NaCl and 3 mM EDTA.
(2) The miniaturized Fc-binding protein after dialysis was converted into HBS-EP + buffer (10 mM HEPES buffer (pH 7.4) containing 150 mM NaCl, 3 mM EDTA, 0.05% (v / v) Surfactant P20). ) To prepare a dilution series from 4.2 nM.
(3) Analyzing the interaction between the Fc-binding protein and human IgG1 using the sensor chip (CM5) on which the 2730.5RU human IgG1 (Sigma-Aldrich) is immobilized using the dilution series prepared in (2) did.

The analysis result is shown in FIG. The miniaturized Fc binding protein has a binding rate constant (ka) of 7.4 × 10 ⁶ (1 / Ms), a dissociation rate constant (kd) of 8.2 × 10 ⁻⁴ (1 / s), and an equilibrium dissociation constant ( KD) is 1.1 × 10 ⁻¹⁰ (M), and has a high binding affinity equivalent to the equilibrium dissociation constant (KD) of wild-type Fc-binding protein: 10 ⁻¹⁰ (M) (Non-patent Document 2). It turned out to have.

Claims (8)

Fc binding protein containing the amino acid from the 7th alanine to the 169th threonine among the amino acid sequences of sequence number 52. An Fc-binding protein comprising at least an amino acid from the 7th alanine to the 169th threonine in the amino acid sequence shown in SEQ ID NO: 52, wherein one or more of the amino acids are substituted with another amino acid. A polynucleotide encoding the amino acid sequence according to claim 1 or 2. A polynucleotide comprising the first to 522th nucleotides in the nucleotide sequence set forth in SEQ ID NO: 7. A vector for expressing an Fc-binding protein, comprising the polynucleotide according to claim 3 or 4. A transformant obtained by transforming a host with the vector according to claim 5. The transformant according to claim 6, wherein the host is Escherichia coli. The manufacturing method of Fc binding protein using the transformant of Claim 6 or 7.

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