JP2015167505A - erythropoietin binding protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an erythropoietin binding protein which has a high affinity to erythropoietin and is capable of efficient production, and to provide a production method thereof.SOLUTION: An erythropoietin binding protein efficiently obtained by culturing a transformant in which Escherichia coli is used as a host has a high affinity to erythropoietin by substituting a specific amino acid into other specific amino acid, among the amino acid which constitutes erythropoietin affinity protein.

Description

  The present invention relates to an erythropoietin-binding protein having binding affinity for erythropoietin. More specifically, the present invention relates to an erythropoietin-binding protein having improved erythropoietin-binding property and production efficiency using genetic engineering techniques.

  Human erythropoietin is a glycoprotein hormone having an action of promoting the differentiation and proliferation of erythroid progenitor cells, and recombinant erythropoietin derived therefrom has already been widely used as a biopharmaceutical such as a therapeutic agent for renal anemia. On the other hand, the protein produced by genetic recombination of the receptor erythropoietin receptor (hereinafter referred to as erythropoietin-binding protein) can also be applied to biopharmaceuticals using its binding ability to erythropoietin. is there. For example, there has been reported a method of treating anemia while preventing hypertension that may occur when an erythropoietin-binding protein is administered together with an erythropoietin preparation to prevent hypertension that may occur when only the erythropoietin preparation is administered to a patient (Patent Document 1). In addition, the use of erythropoietin-binding protein as a therapeutic / improving agent for proliferative organ diseases such as carcinoma, sarcoma, myoma (Patent Document 2), as a prophylactic / therapeutic agent for hypertrophic scar, keloid or chronic arthritic disease. Utilization (Patent Document 3) has also been reported.

  As a production method of erythropoietin-binding protein, a method using an animal cell host (for example, Patent Document 2 and Patent Document 3) or a method using a microbial cell host (for example, Patent Document 1, Patent Document 4 and non-patent document) 1) etc. have been reported.

  However, there is no prior art in which amino acid substitution of erythropoietin-binding protein is performed to improve the productivity and binding to erythropoietin.

Special table 2008-505191 JP-A-10-101574 JP 2002-326958 A JP 2012-147772 A

C. Binnie et al., Protein Expr. Purif. , 11, 271-278, 1997

  An object of the present invention is to provide an erythropoietin-binding protein having high binding ability to erythropoietin and capable of efficient production, and a method for producing the same.

  As a result of intensive studies, the present inventors have found that erythropoietin-binding protein has high binding ability to erythropoietin by substituting a specific amino acid among the amino acids constituting the erythropoietin-binding protein with another specific amino acid. It has been found that the protein can be modified into an erythropoietin-binding protein that can be efficiently produced, and the present invention has been completed.

That is, the present invention
1. The following erythropoietin-binding protein (a) or (b):
(A) In the amino acid sequence represented by SEQ ID NO: 1, the erythropoietin-binding protein comprising the amino acid sequence in which one or more of the following amino acid substitutions (1) to (5) are substituted (1) The 103rd position of SEQ ID NO: 1 Arginine is replaced with serine (2) 141st histidine of SEQ ID NO: 1 is replaced with tyrosine (3) 159th alanine of SEQ ID NO: 1 is replaced with aspartic acid (4) 189th alanine of SEQ ID NO: 1 is aspartic acid Or substituted with valine (5) the 208th cysteine of SEQ ID NO: 1 is substituted with tyrosine (b) in the amino acid sequence of the erythropoietin-binding protein of (a) above, one or more amino acids are deleted, substituted or added 1. An erythropoietin-binding protein consisting of an amino acid sequence. Consisting of the amino acid sequence represented by any one of SEQ ID NO: 2 to SEQ ID NO: 10, The erythropoietin-binding protein according to 1.
3. 1 above. Or 2. A polynucleotide encoding the erythropoietin-binding protein according to any one of the above.
4). 3 above. An expression vector comprising the polynucleotide described in 1.
5. 4. above. A transformant obtained by transforming a host with the expression vector described in 1.
6). 4. The host is E. coli. A transformant according to 1.
7). 5. above. Or 6. A method for producing an erythropoietin-binding protein comprising two steps of producing an erythropoietin-binding protein by culturing the transformant according to claim 1 and recovering the erythropoietin-binding protein produced from the obtained culture. .

  Hereinafter, the present invention will be described in detail.

  The erythropoietin-binding protein of the present invention is a recombinant protein of an erythropoietin receptor, and is a protein that binds to human erythropoietin or a recombinant protein of human erythropoietin.

  The erythropoietin receptor is not particularly limited as long as it is derived from an organism having the erythropoietin receptor, but is preferably a safe human erythropoietin receptor from the viewpoint of industrial use. As an example, the structure of the human erythropoietin receptor will be described. The human erythropoietin receptor consists of 508 amino acids (amino acid sequence of human erythropoietin receptor: registered and published in the UniProt database as accession number P19235), N Signal peptide region consisting of 24 amino acids from the terminal side, extracellular region of erythropoietin receptor consisting of 226 amino acids, transmembrane region of erythropoietin receptor consisting of 23 amino acids, and erythropoietin receptor consisting of 235 amino acids Consists of intracellular regions of the body. Human erythropoietin binds to a portion of the extracellular region of the human erythropoietin receptor.

  The erythropoietin-binding protein of the present invention comprises a substitution of the 103rd arginine of SEQ ID NO: 1 with serine (hereinafter referred to as Arg103Ser) in the amino acid sequence shown in SEQ ID NO: 1, and the 141st histidine of SEQ ID NO: 1. Substitution to tyrosine (hereinafter referred to as His141Tyr), substitution of 159th alanine of SEQ ID NO: 1 to aspartic acid (hereinafter referred to as Ala159Asp), substitution of 189th alanine of SEQ ID NO: 1 to aspartic acid (Hereinafter referred to as Ala189Asp) or a substitution to valine (hereinafter referred to as Ala189Val), and a substitution of the 208th cysteine of SEQ ID NO: 1 to tyrosine (hereinafter referred to as Cys208Tyr). Amino acids with one or more substitutions Column (hereinafter, the amino acid sequence of erythropoietin binding protein of the present invention) erythropoietin binding protein consisting of.

  Describing the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence from the first methionine to the 26th alanine of the amino acid sequence is a MalE signal sequence that is a secretion expression signal to E. coli periplasm, Methionine is a linker sequence, and the amino acid sequence from the 28th alanine to the 252nd aspartic acid is a sequence derived from the amino acid sequence of the extracellular region of the human erythropoietin receptor, and the 258th from the 253rd histidine. The amino acid sequence up to histidine is a polyhistidine sequence.

  As long as the erythropoietin binding protein of the present invention has erythropoietin binding property, one or more amino acids may be deleted, substituted or added in the amino acid sequence of the erythropoietin binding protein of the present invention. Here, the plurality of amino acids means 2 to 170 amino acids, preferably 2 to 50 amino acids, and more preferably 2 to 30 amino acids. For example, a part of the MalE signal sequence which is a secretory expression signal to Escherichia coli periplasm in the amino acid sequence of the erythropoietin-binding protein of the present invention is replaced with another signal sequence (for example, a signal sequence such as PelB, DsbA, TorT, JP 2011-097889 A Publication)) or a portion of the MalE signal sequence may be deleted. Further, for example, the polyhistidine sequence portion in the amino acid sequence of the erythropoietin-binding protein of the present invention may be substituted with other sequences (for example, oligopeptides such as polylysine, polyarginine, polyglutamic acid, polyaspartic acid, etc.). A portion of the polyhistidine sequence may be deleted. Examples of methods for deleting, substituting or adding one or more amino acids in the amino acid sequence of the erythropoietin-binding protein of the present invention include methods using genetic engineering methods well known to those skilled in the art.

  As a specific example of the erythropoietin-binding protein of the present invention, the erythropoietin-binding protein of the present invention comprising the amino acid sequence represented by any of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9 and 10 is used. A protein can be exemplified.

The effects of amino acid substitution of Arg103Ser, His141Tyr, Ala159Asp, Ala189Asp, Ala189Val and Cys208Tyr will be described. The aforementioned His141Tyr is particularly effective in enhancing the erythropoietin binding property of the erythropoietin binding protein of the present invention. The above Arg103Ser, Ala159Asp, Ala189Asp, Ala189Val and Cys208Tyr are particularly effective in increasing the productivity of the erythropoietin-binding protein of the present invention by the expression host.
For example, when the erythropoietin-binding protein of the present invention having a high erythropoietin binding property is desired, it is preferable that the erythropoietin-binding protein of the present invention has the His141Tyr. When the erythropoietin-binding protein of the present invention having high productivity in an expression host is desired, the erythropoietin-binding protein of the present invention is any one or more of Arg103Ser, Ala159Asp, Ala189Asp, Ala189Val and Cys208Tyr. It is preferable to have four amino acid substitutions of Arg103Ser, Ala159Asp, Ala189Asp (or Ala189Val) and Cys208Tyr for higher productivity.

  When the erythropoietin-binding protein of the present invention having high erythropoietin-binding property and high productivity in an expression host is desired, the erythropoietin-binding protein of the present invention has the His141Tyr, and the Arg103Ser, Ala159Asp, Ala189Asp It is preferable to have any one or more of Ala189Val and Cys208Tyr. Furthermore, when the erythropoietin-binding protein of the present invention having a high erythropoietin-binding property and higher productivity in an expression host is desired, the erythropoietin-binding protein of the present invention is the Arg103Ser, His141Tyr, Ala159Asp, Ala189Asp (or Ala189Val). ) And Cys208Tyr with 5 amino acid substitutions.

  Hereinafter, regarding the method for producing the erythropoietin-binding protein of the present invention, production of a polynucleotide encoding the erythropoietin-binding protein of the present invention (hereinafter referred to as the polynucleotide of the present invention), host using the polynucleotide of the present invention And the production of the erythropoietin-binding protein of the present invention using the transformant capable of expressing the erythropoietin-binding protein of the present invention.

  As a method for producing the polynucleotide of the present invention, based on a polynucleotide containing a polynucleotide encoding an erythropoietin receptor, such as an erythropoietin receptor cDNA, a polynucleotide containing a polynucleotide encoding an erythropoietin binding protein, etc. A DNA amplification method such as Chain Reaction (PCR) is used to modify and synthesize the polynucleotide encoding the erythropoietin-binding protein of the present invention, and the amino acid sequence of the erythropoietin-binding protein of the present invention (for example, SEQ ID NO: An example is a method of converting any of 2 to 10) into a polynucleotide sequence and artificially synthesizing a polynucleotide containing the polynucleotide sequence.

In these methods, when converting from an amino acid sequence to a polynucleotide sequence, it is preferable to convert in consideration of the codon usage frequency in the host used for the expression of the erythropoietin-binding protein of the present invention. For example, when the host is E. coli, AGA / AGG / CGG / CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly), proline ( In Pro), since CCC is used less frequently (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). When preparing a polynucleotide using the above DNA amplification method, the polynucleotide of the present invention may be prepared by introducing a mutation using the error-prone PCR method. The reaction conditions in the error-prone PCR method are not particularly limited as long as a desired mutation can be introduced into a polynucleotide encoding an erythropoietin receptor. For example, four types of deoxynucleotides (dATP / dTTP / dCTP / dGTP) concentration is heterogeneous, and MnCl ₂ is added to the PCR reaction solution at a concentration of 0.01 to 10 mM (preferably 0.1 to 1 mM) to introduce a mutation into the polynucleotide. Can do.

  Specific examples of the polynucleotide of the present invention include the polynucleotide of the present invention consisting of the base sequence of SEQ ID NO: 11 encoding the amino acid sequence of SEQ ID NO: 2, the base of SEQ ID NO: 12 encoding the amino acid sequence of SEQ ID NO: 3 A polynucleotide of the present invention comprising the sequence, a polynucleotide of the present invention comprising the base sequence of SEQ ID NO: 13 encoding the amino acid sequence of SEQ ID NO: 4, and a book comprising the base sequence of SEQ ID NO: 14 encoding the amino acid sequence of SEQ ID NO: 5 The polynucleotide of the present invention, the polynucleotide of the present invention consisting of the base sequence of SEQ ID NO: 15 encoding the amino acid sequence of SEQ ID NO: 6, the polynucleotide of the present invention consisting of the base sequence of SEQ ID NO: 16 encoding the amino acid sequence of SEQ ID NO: 7 A book comprising the nucleotide sequence of SEQ ID NO: 17 encoding the amino acid sequence of SEQ ID NO: 8 A polynucleotide of the present invention consisting of the nucleotide sequence of SEQ ID NO: 18 encoding the amino acid sequence of SEQ ID NO: 9, and a polynucleotide of the present invention consisting of the nucleotide sequence of SEQ ID NO: 19 encoding the amino acid sequence of SEQ ID NO: 10 Mention may be made of nucleotides.

  In order to transform a host using the polynucleotide of the present invention, transformation may be performed using the polynucleotide of the present invention itself, but it is usually used for transformation of prokaryotic cells or eukaryotic cells. It is preferable that the polynucleotide of the present invention is inserted at an appropriate position in a bacteriophage, cosmid or plasmid) and transformed using the polynucleotide in view of stable transformation. The expression vector to be used is not particularly limited as long as it stably exists and can replicate in the host to be transformed. For example, as an expression vector when Escherichia coli is used as a host, a pET expression vector, a pUC expression vector, a pTrc expression vector, a pCDF expression vector, a pBBR expression vector and the like can be exemplified.

  The appropriate position means a position where the replication function of the expression vector, a desired antibiotic marker, and a region related to transmissibility are not destroyed. When the polynucleotide of the present invention is inserted into the expression vector, it is preferably inserted into the expression vector in a state of being linked to a functional polynucleotide such as a promoter necessary 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 and the like when the host is Escherichia coli.

  A trait capable of expressing the erythropoietin-binding protein of the present invention by transforming a host using the expression vector inserted with the polynucleotide of the present invention (hereinafter referred to as the expression vector of the present invention) produced by the above method. A transformant (hereinafter referred to as a transformant of the present invention) can be obtained. The host to be transformed is not particularly limited, but Escherichia coli is preferable in terms of easy experiments relating to genetic engineering. In order to transform a host using the expression vector of the present invention, those skilled in the art may carry out the method usually used. For example, when E. coli (E. coli JM109 strain, E. coli BL21 (DE3) strain, E. coli W3110 strain, etc.) is selected as a host, it is described in known literature (for example, Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992). What is necessary is just to transform by a method etc.

  When the expression vector of the present invention is extracted from the transformant of the present invention, it may be extracted using an appropriate extraction method or a commercially available kit. For example, when the host is Escherichia coli, extraction may be performed using an alkaline extraction method or a commercially available extraction kit such as QIAprep Spin Miniprep kit (manufactured by Qiagen).

  In order to produce the erythropoietin-binding protein of the present invention using the transformant of the present invention, the step of producing the erythropoietin-binding protein of the present invention by culturing the transformant of the present invention, and the resulting culture From the above, the production including the two steps of the step of recovering the erythropoietin-binding protein of the present invention may be performed. In the present specification, the culture includes the cultured transformant cells themselves and cell secretions, as well as the medium used for the culture.

  When the transformant of the present invention is cultured, the transformant may be cultured in a medium suitable for host culture. For example, when the host is Escherichia coli, a Luria-Bertani (LB) medium supplemented with necessary nutrients can be mentioned as an example of a preferable medium. In addition, it is preferable to culture by adding a drug corresponding to the drug resistance gene contained in the expression vector of the present invention to the medium because the transformant can be selectively grown depending on whether or not the expression vector of the present invention is introduced. For example, when the expression vector contains a kanamycin resistance gene, kanamycin may be added to the medium.

  In addition to carbon, nitrogen, and inorganic salt sources, a suitable nutrient source may be added to the medium. Furthermore, a reagent such as glycine may be added to the medium to promote protein secretion from the transformant into the culture solution, and the glycine concentration may be in a range that does not affect the growth of E. coli. 10% (W / V) or less, preferably 0.1% to 2%. The culture temperature may be any culture temperature common in the art. For example, when the host is Escherichia coli, the culture temperature is 10 ° C. to 40 ° C., preferably 25 ° C. to 35 ° C., more preferably around 30 ° C. What is necessary is just to select suitably according to the characteristic of the erythropoietin binding protein of this invention to be made. The pH of the medium may be appropriately selected from a general range in the technical field according to various conditions such as the type of the host. For example, when the host is Escherichia coli, the range of pH 6.8 to pH 7.4 is preferable. More preferably, the pH is around 7.0.

  When the expression vector of the present invention contains an inducible promoter, an inducer may be added to induce protein expression under conditions that allow the erythropoietin-binding protein of the present invention to be expressed well. As a preferable inducer, isopropyl-β-D-thiogalactopyranoside (IPTG) can be exemplified. The addition concentration of IPTG may be appropriately selected from the range of 0.005 to 1.0 mM, and is preferably in the range of 0.01 to 0.5 mM. Various conditions relating to IPTG induction may be performed under conditions well known in the art. As a specific example, when the host is Escherichia coli, when the turbidity (absorbance at 600 nm) of the culture solution is about 0.1 to 1.0, an appropriate amount of IPTG is added and then cultured, whereby the erythropoietin of the present invention is obtained. Binding protein expression can be induced.

  In order to recover the erythropoietin-binding protein of the present invention from the culture obtained by culturing the transformant of the present invention, a person skilled in the art may appropriately select and use the method commonly used. The extraction of the erythropoietin-binding protein of the present invention may be carried out according to the form of expression. When secretory production is performed in the culture solution, the cells are separated by centrifugation, and the erythropoietin-binding protein of the present invention may be extracted from the obtained culture supernatant. When expressed in cells (including periplasm in prokaryotes), the cells are collected by centrifugation and then disrupted by adding an enzyme treatment agent or a surfactant, and the cells are removed from the cell lysate. The erythropoietin-binding protein of the invention may be extracted.

  In order to separate and purify the erythropoietin-binding protein of the present invention from the extract obtained by the above-described method, a method known in the art may be used, and as an example, separation and purification using liquid chromatography. Can be mentioned. Examples of liquid chromatography include ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, and the like, and the erythropoietin-binding protein of the present invention can be purified by combining these chromatography methods. Can be recovered with high purity.

An erythropoietin-binding protein having high erythropoietin-binding ability and capable of being efficiently produced, and a method for producing the same are provided.

It is a figure which shows the evaluation result of erythropoietin binding property of erythropoietin binding protein. It is a figure which shows the evaluation result of erythropoietin binding property of erythropoietin binding protein. It is a figure which shows the evaluation result of erythropoietin binding property of erythropoietin binding protein. It is a figure which shows SDS-PAGE of purified EPObp-m5. It is a figure which shows the result of the detection of erythropoietin by ELISA method using EPObp-m5.

  Hereinafter, although an embodiment of the present invention is described in detail using an example, this example is for explaining one form of the present invention and does not limit the present invention.

Example 1 Construction of Expression Vector pETEPObp (1) Construction of pETmalE21 Vector The plasmid vector pETmalEFcR-p7 disclosed in Patent Document 4 as a template DNA, SEQ ID NO: 20 (5′-TAATACGACTCACTATAGGGG-3 ′) and a sequence as PCR primers PCR was performed using oligonucleotides each having the sequence described in No. 21 (5′-TTGTCCCATGGCGAGAGCCGAGGCGGAAAAACAT-3 ′). 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. The obtained PCR product was named MalE21p1.

ＰＣＲ産物ＭａｌＥ２１ｐ１を、アガロースゲルを用いて電気泳動後、目的のＰＣＲ産物を含むゲル部分を切り出し、ＱＩＡｑｕｉｃｋ Ｇｅｌ ｅｘｔｒａｃｔｉｏｎ ｋｉｔ（キアゲン社製）を用いて抽出することで精製した（以下、同様方法での精製をＤＮＡ断片精製と略記する）。精製したＰＣＲ産物ＭａｌＥ２１ｐ１を制限酵素ＸｂａＩとＮｃｏＩで消化後、再度ＤＮＡ断片精製した。制限酵素ＸｂａＩとＮｃｏＩで消化したＰＣＲ産物ＭａｌＥ２１ｐ１を、あらかじめ制限酵素ＸｂａＩとＮｃｏＩで消化したｐＥＴ２６ｂ（＋）ベクター（Ｎｏｖａｇｅｎ社製）へライゲーションすることでｐＥＴＭａｌＥ２１ベクターを作製し、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を塩化カルシウム法により形質転換した。得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養し、ＱＩＡｐｒｅｐ Ｓｐｉｎ Ｍｉｎｉｐｒｅｐ Ｋｉｔ（キアゲン社製）を用いて抽出することでｐＥＴＭａｌＥ２１ベクターを調製した。

The PCR product MalE21p1 was subjected to electrophoresis using an agarose gel, and then the gel portion containing the target PCR product was cut out and purified by extraction using a QIAquick Gel extraction kit (Qiagen) (hereinafter, the same method). Purification is abbreviated as DNA fragment purification). The purified PCR product MalE21p1 was digested with restriction enzymes XbaI and NcoI, and then the DNA fragment was purified again. The PCR product MalE21p1 digested with the restriction enzymes XbaI and NcoI is ligated to the pET26b (+) vector (manufactured by Novagen) previously digested with the restriction enzymes XbaI and NcoI to prepare a pETmalE21 vector, which is used to produce E. coli BL21 ( The DE3) strain was transformed by the calcium chloride method. The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin and extracted using QIAprep Spin Miniprep Kit (manufactured by Qiagen) to prepare pETmalE21 vector.

(2) An erythropoietin-binding protein (hereinafter referred to as wild-type EPObp) expression vector pETEPObp consisting of the amino acid sequence shown in SEQ ID NO: 1 and its transformant were constructed.
(2-1) Plasmid vector pETMalE-p7-EPOR disclosed in Patent Document 4 as template DNA, SEQ ID NO: 22 (5′-TCTCGCCATGGCTCCGCCGCCTAACCTCCCCGGA-3 ′) and SEQ ID NO: 23 (5′-ACACAGCTTCCAAGGCTCATCCCTCGAGCT- PCR was carried out using each of the oligonucleotides having the sequence described in 3 ′). 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. The resulting PCR product was named EPObpp1.

  (2-2) The plasmid vector pETmalE-p7-EPOR disclosed in Patent Document 4 as a template DNA, and SEQ ID NO: 24 (5′-AGCTCGAGGATGAGCCCTTGGAAGCTGTGT-3 ′) and SEQ ID NO: 25 (5′-GCCGCAAGCTTTTAATGATGATGATGATGATGTGTCCA- PCR was carried out using each of the oligonucleotides having the sequence described in 3 ′). 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. The resulting PCR product was named EPObpp2.

  (2-3) After mixing DNA products purified PCR products EPObpp1 and EPObpp2, a reaction solution having the composition shown in Table 2 was prepared. The reaction solution was first step at 98 ° C. for 10 seconds and then at 55 ° C. for 5 seconds. The PCR was carried out by repeating the reaction in which the second step of the above, the third step of 1 minute at 72 ° C. as one cycle, was repeated 5 cycles. The obtained PCR product was named EPObpp3.

（２−４）ＰＣＲ産物ＥＰＯｂｐｐ３を鋳型とし、配列番号２２および配列番号２５に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして、ＰＣＲを実施した。ＰＣＲは、表１に示す組成の反応液を調製後、当該反応液を、９８℃で１０秒間の第１ステップ、５０℃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サイクル繰り返すことで実施した。得られたＰＣＲ産物をＥＰＯｂｐｐ４と命名した。

(2-4) PCR was performed using the PCR product EPObpp3 as a template and oligonucleotides having the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 25 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. The obtained PCR product was named EPObpp4.

  (2-5) After the DNA fragment purified PCR product EPObpp4 is digested with restriction enzymes NcoI and HindIII, the DNA fragment is purified again and ligated to the pETmalE21 vector previously digested with restriction enzymes NcoI and HindIII to produce the expression vector pETEPObp. Using this, Escherichia coli BL21 (DE3) strain was transformed by the calcium chloride method. The obtained transformant (pETEPObp transformant) was cultured in an LB medium supplemented with 50 μg / mL kanamycin, and extracted using QIAprep Spin Miniprep Kit (Qiagen) to prepare an expression vector pETEPObp.

  Of the expression vector pETEPObp, the polynucleotide sequence from the restriction enzyme XbaI recognition sequence to the HindIII recognition sequence is shown in SEQ ID NO: 26.

Example 2 Construction of Random Mutagenesis Library (1) Mutation was randomly introduced into a polynucleotide encoding wild type EPObp using the error-prone PCR method. An expression vector pETEPObp was used as a template DNA, and oligonucleotides having the sequences described in SEQ ID NO: 22 and SEQ ID NO: 25 were used as PCR primers. In error-prone PCR, after preparing a reaction solution having the composition shown in Table 3, the reaction solution is heat-treated at 95 ° C. for 2 minutes, the first step at 95 ° C. for 30 seconds, the second step at 60 ° C. for 30 seconds, 72 The reaction with the third step of 90 seconds at 0 ° C. as one cycle was repeated 30 cycles, and finally heat treatment was carried out at 72 ° C. for 7 minutes.

（２）ＤＮＡ断片精製したＰＣＲ産物を、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化後、再度ＤＮＡ断片精製し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化したｐＥＴＭａｌＥ２１ベクターにライゲーションにより連結し、これを用いてエレクトロポレーション法により大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（３）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを含むＬＢ寒天培地でコロニー形成させることで、エリスロポエチン結合性タンパク質のランダム変異導入ライブラリーを作製した。

(2) The DNA fragment purified PCR product is digested with restriction enzymes NcoI and HindIII, purified again with DNA fragment, ligated to pETmalE21 vector previously digested with restriction enzymes NcoI and HindIII, and electroporated using this. Escherichia coli BL21 (DE3) strain was transformed by the method.
(3) A random mutagenesis library of erythropoietin-binding protein was prepared by colonizing the obtained transformant on an LB agar medium containing 50 μg / mL kanamycin.

Example 3 Screening of erythropoietin-binding protein with high erythropoietin binding (1) Each colony of the random mutagenesis library of erythropoietin-binding protein prepared in Example 2 was treated with LB liquid medium (10 g) containing 50 μg / mL kanamycin. / L Tryptone, 5 g / L Yeast extract, 5 g / L NaCl) was inoculated into 400 μL, and cultured in a 96-well deep well plate at 37 ° C. with aerobic overnight shaking.

(2) After culturing, 20 μL of the culture solution was transferred to 500 μL of LB liquid medium (containing 0.05 mM IPTG, 0.3% glycine, and 50 μg / mL kanamycin), and a 96-well deep well plate was used. The culture was further aerobically shaken at 20 ° C. for 24 hours.
(3) After culture, the culture supernatant obtained by centrifugation was used as an erythropoietin-binding protein sample solution. Each was then diluted 10-fold with TBS-B buffer (20 mM Tris-HCl, 137 mM NaCl, 2.68 mM KCl, 0.5 percent (w / v) Bovine serum albumin, pH 7.4). About the erythropoietin binding protein sample, the binding property with the erythropoietin was evaluated by the enzyme-linked immunosorbent assay (ELISA) method shown below.

(4) Binding evaluation of erythropoietin-binding protein to erythropoietin by ELISA method (4-1) Anti-erythropoietin receptor antibody (Monoclonal Anti-human EPO Antibody) (catalog number MAB3071 manufactured by R & D Systems, Inc., 50 mM) -Adjust to a concentration of 1 μg / mL with HCl buffer (pH 8.0), add it to each well of a 96-well microplate (MaxiSorp, Nunc) at 100 μL / well, and fix the anti-erythropoietin receptor antibody (18 hours at 4 ° C.). After completion of immobilization, the solution in each well was discarded, and TBS-B buffer was added to each well for blocking (30 ° C. for 2 hours).
(4-2) Erythropoietin binding prepared after washing each well with a washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% (w / v) Tween 20 and 150 mM NaCl). Sex protein sample solution was added to the wells and allowed to react with the immobilized antibody (1 hour and a half at 30 ° C).

(4-3) After completion of the reaction, each well was washed with the washing buffer, and erythropoietin (Erythropoietin, Human) which had been biotinylated with EZ-Link Sulfo-NHS-Biotin (manufactured by Thermo scientific) in advance. , Recombinant, CHO Cells, manufactured by Merck Millipore) was adjusted to a concentration of 400 ng / mL with TBS-B buffer and added to each well at 100 μL / well.
(4-4) After reacting at 30 ° C. for 1 hour and a half, each well was washed with the washing buffer, and HRP-Streptavidin conjugate (manufactured by Invitrogen) was diluted 2500 times with TBS-B buffer to each well. Added at 100 μL / well.
(4-5) After reaction for 1 hour and a half at 30 ° C., each well was washed with the washing buffer, TMB Peroxidase Substrate (manufactured by KPL) was added, and the absorbance at 450 nm was measured.

  (5) A transformant expressing an erythropoietin-binding protein exhibiting a high binding property to erythropoietin is selected from about 1200 transformants that have been evaluated by the ELISA method in Example 3 (4). did. As a result, three transformants were selected and designated as A, B and C strains. Each of the selected transformants was cultured, and each expression vector was obtained using QIAprep Spin Miniprep Kit (manufactured by Qiagen).

  (6) Among the obtained expression vectors, a Big Dye Terminator Cycle Sequencing FS read Reaction kit (PE Applied Biosystems) based on the chain terminator method is used for the polynucleotide encoding erythropoietin-binding protein and its surrounding region. The nucleotide sequence was analyzed using a fully automatic DNA sequencer ABI Prism 3700 DNA analyzer (manufactured by PE Applied Biosystems). In the analysis, any one of oligonucleotides having the sequence described in SEQ ID NO: 20 or 27 (5'-TATGCTAGTTATTGCTCAG-3 ') was used as a sequencing primer.

  Based on the results of polynucleotide sequence analysis, the amino acid sequence of the erythropoietin-binding protein expressed by each transformant was determined. As a result, the erythropoietin-binding protein (hereinafter referred to as EPObp-A) expressed by the transformant A strain is His141Tyr (this notation represents substitution of the 141st histidine of SEQ ID NO: 1 with tyrosine, and so on. The erythropoietin-binding protein expressed by the transformant B strain (hereinafter referred to as EPObp-B) is expressed by the transformant C strain with the amino acid substitution of His141Tyr, Asp160Val and Gly190Cys. The erythropoietin-binding protein (hereinafter referred to as EPObp-C) had His97Arg, His141Tyr and Thr220Ala amino acid substitutions, respectively.

  The amino acid sequence of EPObp-A is shown in SEQ ID NO: 2, and the sequence of the polynucleotide encoding the amino acid sequence is shown in SEQ ID NO: 11, respectively. The amino acid sequence of SEQ ID NO: 2 will be described in detail. The amino acid sequence from the first methionine to the 26th alanine of SEQ ID NO: 2 is the MalE signal sequence, the 27th methionine is the linker sequence, and the 28th alanine. To 252nd aspartic acid is a sequence corresponding to the amino acid sequence of the extracellular region of human erythropoietin receptor, and the amino acid sequence from 253rd histidine to 258th histidine is a polyhistidine sequence. The amino acid sequence shown in SEQ ID NO: 2 is an amino acid sequence in which one amino acid is substituted (His141Tyr) in the amino acid sequence shown in SEQ ID NO: 1.

The amino acid sequence of EPObp-B is an amino acid sequence in which two amino acids are further substituted (Asp160Val and Gly190Cys) in the amino acid sequence of SEQ ID NO: 2.
The amino acid sequence of EPObp-C is an amino acid sequence in which two amino acids are further substituted (His97Arg and Thr220Ala) in the amino acid sequence of SEQ ID NO: 2.

Example 4 Evaluation of Transformants Obtained by Screening (1) The transformants A, B and C described in Example 3 were inoculated into an LB liquid medium containing 50 μg / mL kanamycin and incubated at 37 ° C. And pre-cultured by aerobic shaking culture overnight.
(2) The LB liquid medium containing 50 μg / mL kanamycin was inoculated with 1% (v / v) of the preculture, and cultured with shaking at 37 ° C. aerobically.
(3) 1.5 hours after the start of the culture, the culture temperature was changed to 20 ° C., shaking culture for 30 minutes, IPTG was added to a final concentration of 0.01 mM, and then aerobic at 20 ° C. for 22 hours. Cultured with shaking.
(4) After completion of the culture, the cells were collected by centrifugation, and a soluble protein extract was prepared using BugBuster Protein extraction kit (manufactured by Novagen). The concentration of erythropoietin-binding protein in the prepared soluble protein extract was measured using the ELISA method described below.

(5) Quantification of erythropoietin-binding protein by ELISA method (5-1) Anti-erythropoietin receptor antibody (Monoclonal Anti-human EPO Antibody) (manufactured by R & D Systems, catalog number MAB3071) in 50 mM Tris-HCl buffer ( After adjusting the concentration to 1 μg / mL with pH 8.0), it was added to each well of a 96-well microplate (MaxiSorp, Nunc) at 100 μL / well to immobilize the anti-erythropoietin receptor antibody (4 ° C. 18 hours). After immobilization, the solution in each well was discarded, and TBS-B buffer was added to each well for blocking (30 ° C., 2 hours).
(5-2) After blocking, each well was washed with a washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% (w / v) Tween 20 and 150 mM NaCl). Thereafter, the prepared soluble protein extract was serially diluted with TBS-B buffer and reacted with the immobilized antibody in the well (30 ° C., 90 minutes).
(5-3) After washing each well with the washing buffer, Horseradish Peroxidase anti-His-Tag antibody reagent (BETHYL) was added to each well and allowed to react at 30 ° C. for 1.5 hours.

(5-4) After the reaction, each well was washed with the washing buffer, TMB Peroxidase Substrate (manufactured by KPL) was added to each well, and the absorbance at 450 nm was measured.
(5-5) ELISA measurement results of sugar chain-containing erythropoietin-binding protein (Recombinant Human EPO Receptor / EPOR) (manufactured by Sino Biological, catalog number 10707-H08H) (hereinafter referred to as EPObp with sugar chain) From the measured absorbance, the protein concentration of erythropoietin-binding protein in the soluble protein extract and the production amount of erythropoietin-binding protein per culture solution were determined. The amino acid sequence of EPObp with sugar chain is the polyhistidine sequence from the first methionine to the 250th proline of the amino acid sequence of human erythropoietin receptor (registered and published in the UniProt database as accession number P19235). Consists of an amino acid sequence appended to the C-terminus. The amino acid sequence from the first methionine to the 24th tryptophan in the amino acid sequence of this EPObp with sugar chain is the sequence of the signal peptide region, and this signal peptide region is lost in the mature EPObp with sugar chain after expression.

(6) The soluble protein extract prepared in (4) of Example 4 was diluted with TBS-B buffer so that the concentration of erythropoietin-binding protein was 400 ng / mL. Using this, the binding property of each erythropoietin-binding protein to erythropoietin was evaluated by the ELISA method described in (3) of Example 3.
(7) As a comparative control, the same experiment as that described in (1) to (6) of Example 4 was performed for the pETEPObp transformant described in Example 1 capable of expressing wild-type EPObp. Further, as a comparative control, an experiment similar to the experiment described in (5) and (6) of Example 4 was performed for EPObp with a sugar chain.

  (8) As a result of performing the measurement in duplicate, the amount of erythropoietin-binding protein per culture solution by each transformant was, in descending order, the transformant A strain (EPObp-A production amount per culture solution: 3.9 ± 0.7 mg / L), pETEPObp transformant (production amount of wild type EPObp per culture solution: 1.6 ± 0.2 mg / L), transformant C strain (EPObp− per culture solution) C production: 0.5 ± 0.0 mg / L), and transformant B strain (production amount of EPObp-B per culture solution: 0.4 ± 0.0 mg / L).

  (9) The results of evaluating the binding properties of each erythropoietin-binding protein to erythropoietin are shown in FIG. The vertical axis | shaft of FIG. 1 is the value of the light absorbency in 450 nm of ELISA, and shows that erythropoietin binding property is so high that it is large. Measurements were performed in duplicate. Since E. coli transformants are used, no sugar chain is added to the erythropoietin-binding protein produced by each transformant.

  As shown in FIG. 1, the erythropoietin binding property of wild-type EPObp was lower than that of EPObp with a sugar chain. The remaining amino acid sequence portion excluding the MalE signal sequence of the amino acid sequence of wild-type EPObp (the amino acid sequence portion from the 27th methionine to the 258th histidine of the amino acid sequence shown in SEQ ID NO: 1) is almost the same as the EPObp with sugar chain The same amino acid sequence. From this, it was speculated that wild-type EPObp has low erythropoietin-binding properties because there is no sugar chain. This is because erythropoietin binding affinity (dissociation constant KD = 2.9 nM) of an erythropoietin-binding protein having no sugar chain, which is reported in a known literature, is the same as that of an erythropoietin-binding protein having a sugar chain. Supported by lower erythropoietin binding affinity (dissociation constant KD = 0.13 nM) (S. Liu et al., Proc. Natl. Acad. Sci. USA, 104, 5330-5335, 2007) .

  On the other hand, EPObp-A, EPObp-B, and EPObp-C all have no sugar chain, but showed higher erythropoietin-binding properties than wild-type EPObp (FIG. 1). That is, from this result, it was suggested that the substitution of amino acids possessed by each of EPObp-A, EPObp-B and EPObp-C contributed to the improvement of erythropoietin binding. EPObp-A, EPObp-B and EPObp-C all had His141Tyr amino acid substitution, and EPObp-A which had only His141Tyr as an amino acid substitution had high erythropoietin binding. From these results, it was suggested that amino acid substitution of His141Tyr is important for improving erythropoietin binding.

Example 5 Construction of Random Mutagenesis Library (1) Mutations were randomly introduced into a polynucleotide encoding EPObp-A using the error-prone PCR method. An expression vector obtained from the transformant A strain described in (5) of Example 3 was used as a template DNA, and oligonucleotides having the sequences described in SEQ ID NO: 22 and SEQ ID NO: 25 were used as PCR primers. In error-prone PCR, after preparing a reaction solution having the composition shown in Table 3, the reaction solution is heat-treated at 95 ° C. for 2 minutes, the first step at 95 ° C. for 30 seconds, the second step at 60 ° C. for 30 seconds, 72 The reaction with the third step of 90 seconds at 0 ° C. as one cycle was repeated 30 cycles, and finally heat treatment was carried out at 72 ° C. for 7 minutes.

(2) The DNA fragment purified PCR product is digested with restriction enzymes NcoI and HindIII, purified again with DNA fragment, ligated to pETmalE21 vector previously digested with restriction enzymes NcoI and HindIII, and electroporated using this. Escherichia coli BL21 (DE3) strain was transformed by the method.
(3) A random mutagenesis library of erythropoietin-binding protein was prepared by colonizing the obtained transformant on an LB agar medium containing 50 μg / mL kanamycin.

Example 6 Screening for Erythropoietin Binding Protein (1) Each colony of the random mutagenesis library prepared in Example 5 was inoculated into 400 μL of LB liquid medium containing 50 μg / mL kanamycin, and a 96-well deep well plate was used. The culture was shaken aerobically overnight at 37 ° C.
(2) After culturing, 20 μL of the culture solution was transferred to 500 μL of LB liquid medium (containing 0.05 mM IPTG, 0.3% glycine, and 50 μg / mL kanamycin), and a 96-well deep well plate was used. The culture was further aerobically shaken at 20 ° C. for 24 hours.

(3) After culture, the culture supernatant obtained by centrifugation was used as an erythropoietin-binding protein sample solution. In order to enhance the selectivity during screening, the following citrate buffer treatment was performed on the erythropoietin-binding protein sample solution. 20 μL of each erythropoietin binding protein sample solution was mixed with 30 μL of 0.1 M citrate buffer (pH 3.0) and placed at 30 ° C. for 1 hour. Thereafter, 25 μL of 1 M Tris-HCl buffer (pH 7.4) was further mixed with this mixed solution to obtain a citrate buffer treated sample solution.
(4) The citrate buffer-treated sample solution of (3) of Example 6 was diluted 2-fold with TBS-B buffer, and the erythropoietin binding property of the erythropoietin-binding protein contained therein was changed to (4) of Example 3. ) Was evaluated by ELISA.

  (5) The erythropoietin-binding property that showed high binding property to erythropoietin even after the citrate buffer treatment from among about 1200 transformants that were evaluated by the ELISA method in Example 6 (4). A transformant expressing the protein was selected. As a result, four transformants were selected and named D strain, E strain, F strain and G strain. Each of the selected transformants was cultured, and an expression vector was obtained using QIAprep Spin Miniprep Kit.

  (6) Among the obtained expression vectors, the polynucleotide encoding the erythropoietin-binding protein and the nucleotide sequence of the surrounding region were analyzed by the same method as described in (3) of Example 3. .

  As a result of examining the amino acid sequence based on the sequence of the polynucleotide, the erythropoietin-binding protein (hereinafter referred to as EPObp-D) expressed by the transformant D strain has an amino acid substitution of Ala189Asp in addition to His141Tyr. (The amino acid sequence of EPObp-D is shown in SEQ ID NO: 3, and the sequence of the polynucleotide encoding the amino acid sequence is shown in SEQ ID NO: 12, respectively). The erythropoietin-binding protein (hereinafter referred to as EPObp-E) expressed by the transformant E strain has Arg103Ser and Ala189Val amino acid substitutions in addition to His141Tyr (the amino acid sequence of EPObp-E is represented by SEQ ID NO: 4). The sequence of the polynucleotide encoding the amino acid sequence is shown in SEQ ID NO: 13, respectively).

  The erythropoietin-binding protein (hereinafter referred to as EPObp-F) expressed by the transformant F strain had an amino acid substitution of Ala159Asp in addition to His141Tyr (the amino acid sequence of EPObp-F in SEQ ID NO: 5). The sequence of the polynucleotide encoding the amino acid sequence is shown in SEQ ID NO: 14, respectively). The erythropoietin-binding protein (hereinafter referred to as EPObp-G) expressed by the transformant G strain has a substitution of amino acids of Cys208Tyr in addition to His141Tyr (the amino acid sequence of EPObp-G is shown in SEQ ID NO: 6, The sequence of the polynucleotide encoding the amino acid sequence is shown in SEQ ID NO: 15).

  (7) For the transformant D strain, E strain, F strain and G strain described in (5) of Example 6, the same experiment as that described in (1) to (6) of Example 4 was performed. The amount of erythropoietin-binding protein produced by each transformant and the erythropoietin-binding property of the produced erythropoietin-binding protein were evaluated.

  As a comparative control, the same experiment as that described in (1) to (6) of Example 4 was performed for the transformant A strain described in Example 3. Further, as a comparative control, an experiment similar to the experiment described in (5) and (6) of Example 4 was performed for EPObp with a sugar chain.

  As a result of measurement in duplicate, the amount of erythropoietin-binding protein per culture broth by each transformant was in descending order of transformant E strain (production amount of EPObp-E per culture broth: 8.7). ± 0.1 mg / L), transformant D strain (production amount of EPObp-D per culture broth: 6.6 ± 0.8 mg / L), transformant G strain (EPObp-G per culture broth) Production amount: 5.2 ± 1.7 mg / L), transformant F strain (production amount of EPObp-F per culture solution: 4.8 ± 0.8 mg / L), transformant A strain (culture solution) Per EPObp-A production: 1.1 ± 0.2 mg / L). From these results, it was suggested that substitution of amino acids other than His141Tyr, which each of EPObp-D, EPObp-E, EPObp-F and EPObp-G had, contributed to an improvement in the production of erythropoietin-binding protein. It was.

  The results of evaluating the binding of each erythropoietin-binding protein to erythropoietin are shown in FIG. The vertical axis | shaft of FIG. 2 is the value of the light absorbency in 450 nm of ELISA, and shows that erythropoietin binding property is so high that it is large. Measurements were performed in duplicate. Since E. coli transformants are used, no sugar chain is added to the erythropoietin-binding protein produced by each transformant. As shown in FIG. 2, EPObp-D, EPObp-E, EPObp-F and EPObp-G all have no sugar chain, but showed a high erythropoietin-binding property higher than EPObp-A. From these results, it was suggested that substitution of amino acids other than His141Tyr of each of EPObp-D, EPObp-E, EPObp-F and EPObp-G contributed to the improvement of erythropoietin binding.

Example 7 Production of erythropoietin binding protein EPObp-m3a Cys208Tyr was selected from the amino acid substitutions found in Example 6, and this amino acid substitution was compared to erythropoietin binding protein EPObp-D described in Example 6. Furthermore, erythropoietin-binding protein EPObp-m3a having three amino acid substitutions (His141Tyr, Ala189Asp, and Cys208Tyr) was produced by introduction.

(1) The following PCR was performed to further introduce amino acid substitution of Cys208Tyr.
(1-1) An EPObp-D expression vector prepared from the transformant D strain in Example 6 as a template, and a sequence described in SEQ ID NO: 20 and SEQ ID NO: 28 (5'-AGGTTGCTCAGCACACTACTGGTGCG-3 ') as a PCR primer PCR was carried out using each of the oligonucleotides consisting of 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. The obtained PCR product was named m3ap1.
(1-2) The expression vector of EPObp-D prepared from the transformant D strain in Example 6 as a template, SEQ ID NO: 29 (5′-CGCACCGAGGATGTGCTGAGCAACCT-3 ′) and the sequence described in SEQ ID NO: 27 as PCR primers PCR was carried out using each of the oligonucleotides consisting of 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. The obtained PCR product was named m3ap2.

(1-3) After mixing the DNA fragments purified PCR products m3ap1 and m3ap2, prepare a reaction solution having the composition shown in Table 2 and prepare the reaction solution at 98 ° C. for 10 seconds in the first step and 55 ° C. for 5 seconds. The PCR was carried out by repeating the reaction in which the second step of the above, the third step of 1 minute at 72 ° C. as one cycle, was repeated 5 cycles. The obtained PCR product was named m3ap3.
(1-4) PCR was carried out using the PCR product m3ap3 as a template and oligonucleotides having the sequences shown in SEQ ID NO: 20 and SEQ ID NO: 27 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. The obtained PCR product was purified with a DNA fragment to obtain a polynucleotide encoding erythropoietin-binding protein EPObp-m3a.

(2) Polynucleotide encoding erythropoietin-binding protein EPObp-m3a is digested with restriction enzymes NcoI and HindIII, DNA fragment purified, ligated to pETmalE21 vector previously digested with restriction enzymes NcoI and HindIII by ligation, and used Escherichia coli BL21 (DE3) strain was transformed by the calcium chloride method.
(3) The obtained transformant was named transformant m3a strain. This transformant m3a strain is cultured in an LB medium supplemented with 50 μg / mL kanamycin, and an expression vector is extracted to obtain an expression vector pETEPObp-m3a containing a polynucleotide encoding erythropoietin-binding protein EPObp-m3a. It was.

  (4) In the expression vector pETEPObp-m3a, the analysis of the sequence of the polynucleotide region encoding the erythropoietin-binding protein EPObp-m3a was performed by the same method as (6) of Example 3.

  The amino acid sequence of the polypeptide expressed by the expression vector pETEPObp-m3a is shown in SEQ ID NO: 7, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 16, respectively. The amino acid sequence of SEQ ID NO: 7 will be described in detail. The amino acid sequence from the first methionine to the 26th alanine of SEQ ID NO: 7 is the MalE signal sequence, the 27th methionine is the linker sequence, and the 28th alanine. To 252nd aspartic acid is a sequence corresponding to the amino acid sequence of the extracellular region of human erythropoietin receptor, and the amino acid sequence from 253rd histidine to 258th histidine is a polyhistidine sequence. The amino acid sequence shown in SEQ ID NO: 7 is an amino acid sequence in which three amino acids are substituted in the amino acid sequence shown in SEQ ID NO: 1 (His141Tyr, Ala189Asp, and Cys208Tyr).

Example 8 Production of erythropoietin binding protein EPObp-m3b Ala159Asp was selected from the amino acid substitutions found in Example 6, and this amino acid substitution was compared to erythropoietin binding protein EPObp-D described in Example 6. Furthermore, erythropoietin-binding protein EPObp-m3b having three amino acid substitutions (His141Tyr, Ala159Asp and Ala189Asp) was prepared by introduction.

(1) The following PCR was performed to further introduce amino acid substitution of Ala159Asp.
(1-1) An EPObp-D expression vector prepared from the transformant D strain in Example 6 as a template, and a sequence described in SEQ ID NO: 20 and SEQ ID NO: 30 (5′-TCTCGTCATCCCAACCCGGCCCACCA-3 ′) as a PCR primer PCR was carried out using each of the oligonucleotides consisting of 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. The obtained PCR product was named m3bp1.
(1-2) The expression vector of EPObp-D prepared from the transformant D strain in Example 6 as a template, SEQ ID NO: 31 (5′-TGGTGGCGCGGGTGGATGGAGAGA-3 ′) and the sequence described in SEQ ID NO: 27 as PCR primers PCR was carried out using each of the oligonucleotides consisting of 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. The obtained PCR product was named m3bp2.

(1-3) After mixing the DNA fragments purified PCR products m3bp1 and m3bp2, a reaction solution having the composition shown in Table 2 is prepared. The reaction solution is first step at 98 ° C. for 10 seconds, and 5 seconds at 55 ° C. The PCR was carried out by repeating the reaction in which the second step of the above, the third step of 1 minute at 72 ° C. as one cycle, was repeated 5 cycles. The obtained PCR product was named m3bp3.
(1-4) PCR was performed using the PCR product m3bp3 as a template and oligonucleotides having the sequences shown in SEQ ID NO: 20 and SEQ ID NO: 27 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. The obtained PCR product was purified with a DNA fragment to obtain a polynucleotide encoding erythropoietin-binding protein EPObp-m3b.

(2) Polynucleotide encoding erythropoietin-binding protein EPObp-m3b is digested with restriction enzymes NcoI and HindIII, DNA fragment purified, ligated to pETmalE21 vector previously digested with restriction enzymes NcoI and HindIII by ligation and used Escherichia coli BL21 (DE3) strain was transformed by the calcium chloride method.
(3) The obtained transformant was named transformant m3b strain. This transformant m3b strain is cultured in an LB medium supplemented with 50 μg / mL kanamycin, and an expression vector is extracted to obtain an expression vector pETEPObp-m3b containing a polynucleotide encoding erythropoietin-binding protein EPObp-m3b. It was.

  (4) In the expression vector pETEPObp-m3b, the sequence of the polynucleotide region encoding the erythropoietin-binding protein EPObp-m3b was analyzed by the same method as in Example 6, (6).

  The amino acid sequence of the polypeptide expressed by the expression vector pETEPObp-m3b is shown in SEQ ID NO: 8, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 17, respectively. The amino acid sequence of SEQ ID NO: 8 will be described in detail. The amino acid sequence from the first methionine to the 26th alanine of SEQ ID NO: 8 is a MalE signal sequence, the 27th methionine is a linker sequence, and the 28th alanine. To the 252nd aspartic acid is a sequence corresponding to the amino acid sequence of the extracellular region of the human erythropoietin receptor, and the amino acid sequence from the 253rd histidine to the 258th histidine is a polyhistidine sequence. The amino acid sequence shown in SEQ ID NO: 8 is an amino acid sequence in which three amino acids are substituted in the amino acid sequence shown in SEQ ID NO: 1 (His141Tyr, Ala159Asp and Ala189Asp).

Example 9 Production of erythropoietin-binding protein EPObp-m4 Ala159Asp and Cys208Tyr were selected from the amino acid substitutions identified in Example 6, and these amino acid substitutions were performed using erythropoietin-binding protein EPObp-D described in Example 6. Was further introduced to produce erythropoietin-binding protein EPObp-m4 having four amino acid substitutions (His141Tyr, Ala159Asp, Ala189Asp, and Cys208Tyr).

(1) The following PCR was performed to further introduce amino acid substitutions of Ala159Asp and Cys208Tyr.
(1-1) PCR was performed using the expression vector pETEPObp-m3a of Example 7 as a template and oligonucleotides having the sequences described in SEQ ID NO: 31 and SEQ ID NO: 27 as PCR primers, respectively. 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. The obtained PCR product was named m4p1.

(1-2) After the DNA fragment purified PCR product m4p1 and the PCR product m3bp1 described in Example 1-1 (1-1) were mixed, a reaction solution having the composition shown in Table 2 was prepared. PCR was carried out by repeating 5 cycles of a reaction in which the first step at 10 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, and the third step at 72 ° C. for 1 minute was 1 cycle. The obtained PCR product was named m4p2.
(1-3) PCR was performed using the PCR product m4p2 as a template and oligonucleotides having the sequences shown in SEQ ID NO: 20 and SEQ ID NO: 27 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. The obtained PCR product was purified with a DNA fragment to obtain a polynucleotide encoding erythropoietin-binding protein EPObp-m4.

(2) Polynucleotide encoding erythropoietin-binding protein EPObp-m4 is digested with restriction enzymes NcoI and HindIII, DNA fragment purified, ligated to pETmalE21 vector previously digested with restriction enzymes NcoI and HindIII by ligation, and used Escherichia coli BL21 (DE3) strain was transformed by the calcium chloride method.
(3) The obtained transformant was named transformant m4 strain. This transformant m4 strain is cultured in an LB medium supplemented with 50 μg / mL kanamycin, and an expression vector is extracted to obtain an expression vector pETEPObp-m4 containing a polynucleotide encoding erythropoietin-binding protein EPObp-m4. It was.

  (4) In the expression vector pETEPObp-m4, the sequence of the polynucleotide region encoding erythropoietin-binding protein EPObp-m4 was analyzed by the same method as (6) of Example 3.

  The amino acid sequence of the polypeptide expressed by the expression vector pETEPObp-m4 is shown in SEQ ID NO: 9, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 18, respectively. The amino acid sequence of SEQ ID NO: 9 will be described in detail. The amino acid sequence from the first methionine to the 26th alanine of SEQ ID NO: 9 is a MalE signal sequence, the 27th methionine is a linker sequence, and the 28th alanine. To 252nd aspartic acid is a sequence corresponding to the amino acid sequence of the extracellular region of human erythropoietin receptor, and the amino acid sequence from 253rd histidine to 258th histidine is a polyhistidine sequence. The amino acid sequence shown in SEQ ID NO: 9 is an amino acid sequence in which four amino acids are substituted in the amino acid sequence shown in SEQ ID NO: 1 (His141Tyr, Ala159Asp, Ala189Asp, and Cys208Tyr).

Example 10 Production of erythropoietin-binding protein EPObp-m5 Arg103Ser was selected from the amino acid substitutions found in Example 6, and this amino acid substitution was performed on erythropoietin-binding protein EPObp-m4 produced in Example 9. Furthermore, erythropoietin-binding protein EPObp-m5 having five amino acid substitutions (Arg103Ser, His141Tyr, Ala159Asp, Ala189Asp and Cys208Tyr) was prepared by introduction.

(1) The following PCR was performed to further introduce Arg103Ser amino acid substitution.
(1-1) Using the expression vector pETEPObp-m4 of Example 9 as a template and oligonucleotides having the sequences described in SEQ ID NO: 20 and SEQ ID NO: 32 (5′-ACCGCACCACTAGCCGTGGGAGCCT-3 ′) as PCR primers, respectively. PCR was performed. 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. The resulting PCR product was named m5p1.

  (1-2) Using the expression vector pETEPObp-m4 of Example 9 as a template and oligonucleotides having the sequences described in SEQ ID NO: 33 (5′-AGGCTCCCACGGCTAGTGGTGCGGGT-3 ′) and SEQ ID NO: 27 as PCR primers, respectively. PCR was performed. 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. The obtained PCR product was named m5p2.

(1-3) After mixing the DNA products purified PCR products m5p1 and m5p2, a reaction solution having the composition shown in Table 2 was prepared, and the reaction solution was first step at 98 ° C. for 10 seconds, and then at 55 ° C. for 5 seconds. The PCR was carried out by repeating the reaction in which the second step of the above, the third step of 1 minute at 72 ° C. as one cycle, was repeated 5 cycles. The obtained PCR product was named m5p3.
(1-4) PCR was performed using the PCR product 5p3 as a template and oligonucleotides having the sequences described in SEQ ID NO: 20 and SEQ ID NO: 27 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 erythropoietin-binding protein EPObp-m5 was obtained.

(2) Polynucleotide encoding erythropoietin-binding protein EPObp-m5 is digested with restriction enzymes NcoI and HindIII, DNA fragment purified, ligated to pETmalE21 vector previously digested with restriction enzymes NcoI and HindIII by ligation and used Escherichia coli BL21 (DE3) strain was transformed by the calcium chloride method.
(3) The obtained transformant was named transformant m5 strain. This transformant m5 strain is cultured in an LB medium supplemented with 50 μg / mL kanamycin, and an expression vector is extracted to obtain an expression vector pETEPObp-m5 containing a polynucleotide encoding erythropoietin-binding protein EPObp-m5. It was.

  (4) In the expression vector pETEPObp-m5, the sequence of the polynucleotide region encoding the erythropoietin-binding protein EPObp-m5 was analyzed by the same method as (6) of Example 3.

  The amino acid sequence of the polypeptide expressed by the expression vector pETEPObp-m5 is shown in SEQ ID NO: 10, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 19, respectively. The amino acid sequence of SEQ ID NO: 10 will be described in detail. The amino acid sequence from the first methionine to the 26th alanine of SEQ ID NO: 10 is a MalE signal sequence, the 27th methionine is a linker sequence, and the 28th alanine. To 252nd aspartic acid is a sequence corresponding to the amino acid sequence of the extracellular region of human erythropoietin receptor, and the amino acid sequence from 253rd histidine to 258th histidine is a polyhistidine sequence. The amino acid sequence shown in SEQ ID NO: 10 is an amino acid sequence in which five amino acids are substituted in the amino acid sequence shown in SEQ ID NO: 1 (Arg103Ser, His141Tyr, Ala159Asp, Ala189Asp, and Cys208Tyr).

Example 11 Evaluation of Erythropoietin Binding Protein (1) Transformant A strain of Example 3, Transformant D strain of Example 6, Transformant m3a strain of Example 7, Transformant m3b of Example 8 Strain, the transformant m4 strain of Example 9 and the transformant m5 strain of Example 10 were each inoculated into LB liquid medium containing 50 μg / mL kanamycin and aerobically shaken overnight at 37 ° C. Pre-culture was performed by culturing.
(2) Each preculture was inoculated with 1% (v / v) of LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37 ° C. aerobically.
(3) 1.5 hours after the start of culture, change the culture temperature to 20 ° C., shake culture for 30 minutes, then add IPTG to a final concentration of 0.01 mM, and continue at 20 ° C. for 22 hours. The culture was shaken vigorously.

(4) After culturing, the cells were collected by centrifugation, and each soluble protein extract was prepared using BugBuster Protein extraction kit (Novagen).
(5) The concentration of erythropoietin-binding protein in the soluble protein extract prepared in (4) of Example 11 was measured by the same method as the ELISA method described in (4) of Example 4.
(6) The soluble protein extract prepared in (4) of Example 11 was diluted with TBS-B buffer so that the concentration of each erythropoietin-binding protein was 375 ng / mL. Using these, the binding property of each erythropoietin-binding protein to erythropoietin was evaluated by the ELISA method described in (3) of Example 3.
(7) The same experiment as that described in (1) to (6) of Example 11 was performed for the pETEPObp transformant described in Example 1 capable of expressing wild type EPObp as a comparative control. As a comparative control, an experiment similar to the experiment described in (5) and (6) of Example 11 was performed for EPObp with a sugar chain.

  (8) As a result of measuring in duplicate, the production amount of erythropoietin-binding protein per culture broth by each transformant was in descending order of the transformant m5 strain (production amount of EPObp-m5: 210.7 ± 22.7 mg / L), transformant m4 strain (production amount of EPObp-m4: 70.9 ± 10.3 mg / L), transformant m3a strain (production amount of EPObp-m3a: 33.3 ± 3. 2 mg / L), transformant m3b strain (production amount of EPObp-m3b: 24.2 ± 0.6 mg / L), transformant D strain (production amount of EPObp-D: 8.2 ± 2.0 mg / L) L), transformant A strain (EPObp-A production: 2.6 ± 0.3 mg / L), pETEPObp transformant (wild-type EPObp production: 2.3 ± 0.1 mg / L) there were. Thus, it can be seen that the production amount of erythropoietin-binding protein is improved by having a specific amino acid substitution. And it turns out that the production amount improves more because erythropoietin binding protein has many substitution of a specific amino acid.

  (9) The results of evaluating the binding of each erythropoietin-binding protein to erythropoietin are shown in FIG. The vertical axis | shaft of FIG. 3 is the value of the light absorbency in 450 nm of ELISA, and shows that erythropoietin binding property is so high that it is large. Measurements were performed in duplicate. As shown in FIG. 3, wild-type EPObp has low erythropoietin-binding property and could not be detected. On the other hand, EPObp-A, EPObp-D, EPObp-m3a, EPObp-m3b, EPObp-m4, and EPObp-m5 all show higher erythropoietin-binding properties compared to wild-type EPObp, and erythropoietin is equal to or higher than EPObp with sugar chains. Showed binding.

Example 12 Analysis of erythropoietin binding affinity of EPObp-m5 (1) EPObp-m5 is expressed in the transformant m5 strain of Example 10 in the same manner as in the methods of (1) to (4) of Example 11. A soluble protein extract containing EPObp-m5 was prepared.
(2) Using HIS-Select Cobalt Affinity Gel (Sigma-Aldrich), EPObp-m5 having a histidine tag was purified from the soluble protein extract containing EPObp-m5 prepared in (1) of Example 12. A purified solution was obtained. The purification method was performed according to the recommended method of HIS-Select Cobalt Affinity Gel.

(3) The purified solution is mixed with a sample buffer for sodium dodecylsulfide gel gel electrophoresis (SDS-PAGE) (Easy Apply, manufactured by ATTO), then heat-denatured at 98 ° C. for 10 minutes, and 15% polyacrylamide gel SDS-PAGE was performed using (e-PAGE, manufactured by Atto Corporation). At this time, Prestained protein marker, broad range (manufactured by New England Biolabs) was used as a molecular weight marker. After electrophoresis, the protein in the polyacrylamide gel was detected using Easy Stain Aqua (manufactured by Atto).
The results of this SDS-PAGE are shown in FIG. As shown in the result of SDS-PAGE in FIG. 4, EPObp-m5 of the purified solution was observed as a single band at the position of the size estimated from the amino acid sequence (about 25.8 kDa, not including the MalE signal portion). In addition, since other bands were not observed, it was confirmed that the purity was high.
(4) The concentration of EPObp-m5 in the purified solution was determined from the absorbance at 280 nm.

(5) Using Biacore T100 (manufactured by GE Healthcare), analysis by surface plasmon resonance was performed on the binding between EPObp-m5 and erythropoietin. The experiment was performed according to the recommended method of Biacore T100. As the running buffer, HBS-EP + (manufactured by GE Healthcare) was used.
(5-1) EPObp-m5 was immobilized on 283.8RU in Sensor Chip CM5 (manufactured by GE Healthcare). An amine coupling kit (manufactured by GE Healthcare) was used for immobilization at this time.

(5-2) A solution of 10 nM erythropoietin (Erythropoietin, Human, Recombinant, CHO Cells, manufactured by Merck Millipore) was diluted stepwise to 0.3 nM with HBS-EP + at a 2-fold dilution. Each concentration of erythropoietin solution was passed through Sensor Chip CM5 in which EPObp-m5 of Example 5-1 (5-1) was immobilized at 25 ° C. at a flow rate of 30 μL / second for 210 seconds. Regeneration of Sensor Chip CM5 immobilized with EPObp-m5 was performed using 10 mM glycine buffer (pH 2.5).
(5-3) The association rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD) were 1: 1 with BIAevaluation software (version 1.1.1) (manufactured by GE Healthcare). It was determined from the results of curve fitting analysis using a binding model.

(5-4) a series of analysis was performed in duplicate, the values of Rmax is 81.6 or 78.6RU, squared chi value was 0.47 or 0.32RU ^2. As a result of the analysis, the binding rate constant (ka) of the binding between EPObp-m5 and erythropoietin is (1.4 ± 0.0) × 10 ⁶ M ⁻¹ s ⁻¹ , and the dissociation rate constant (kd) is (1.3 ± 0.0) × 10 ⁻⁴ s ⁻¹ and the dissociation constant (KD) was 0.09 ± 0.00 nM.
The value of dissociation constant (KD) in erythropoietin binding affinity reported in known literature is KD = 2.9 nM for erythropoietin-binding protein without sugar chain (Non-patent Document 1), and erythropoietin with sugar chain The binding protein is KD = 0.13 nM (S. Liu et al., Proc. Natl. Acad. Sci. USA, 104, 5330-5335, 2007). Compared with these dissociation constant values, the dissociation constant value of erythropoietin binding affinity of EPObp-m5 is as small as KD = 0.09 ± 0.00 nM, so that EPObp-m5 has high erythropoietin binding affinity. I understand.

Example 13 Detection of erythropoietin using EPObp-m5 (1) EPObp-m5 was expressed in the transformant m5 strain of Example 10 in the same manner as in methods (1) to (4) of Example 11. A soluble protein extract containing EPObp-m5 was prepared.
(2) Using HIS-Select Nickel Affinity Gel (manufactured by Sigma-Aldrich), EPObp-m5 having a histidine tag was purified from the soluble protein extract containing EPObp-m5 prepared in (1) of Example 13. A purified solution was obtained. The purification method was performed according to the recommended method of HIS-Select Nickel Affinity Gel.
(3) The concentration of EPObp-m5 in the purified solution prepared in (2) of Example 13 was measured by the same method as the ELISA method described in (5) of Example 4.

(4) Detection of erythropoietin by ELISA using EPObp-m5 was performed by the following method.
(4-1) Anti-erythropoietin receptor antibody (Monoclonal Anti-human Epoxy Antibody) (manufactured by R & D Systems, catalog number MAB3071) was adjusted to a concentration of 1 μg / mL with 50 mM Tris-HCl buffer (pH 8.0). Thereafter, it was added to each well of a 96-well microplate (MaxiSorp, Nunc) at 100 μL / well to immobilize the anti-erythropoietin receptor antibody (18 ° C. for 18 hours). After immobilization, the solution in each well was discarded, and TBS-B buffer was added to each well for blocking (30 ° C., 2 hours). After blocking, each well was washed with a washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% (w / v) Tween 20 and 150 mM NaCl).
(4-2) EPObp-m5 purified in (13) of Example 13 was adjusted to a concentration of 400 ng / mL with TBS-B buffer, and added to each well at 100 μL / well. After 1 hour and a half reaction at 30 ° C., each well was washed with the washing buffer.

(4-3) Erythropoietin (Erythropoietin, Human, Recombinant, CHO Cells, manufactured by Merck Millipore) in TBS-B buffer at various concentrations (6 ng / mL, 12 ng / mL, 24 ng / mL, 49 ng / mL, 98 ng / mL) And 196 ng / mL), and added to each of three wells at 100 μL / well. After 1 hour and a half reaction at 30 ° C., each well was washed with the washing buffer.
(4-4) Anti-erythropoietin antibody (Anti-Erythropoietin, Mouse-Mono (Epo1)) manufactured by EZ-Link Sulfo-NHS-Biotin (manufactured by Thermo scientific) , Catalog number AM00744PU-N) was adjusted to a concentration of 3.8 μg / mL with TBS-B buffer and added to each well at 100 μL / well. After 1 hour and a half reaction at 30 ° C., each well was washed with the washing buffer.
(4-5) HRP-Streptavidin conjugate (manufactured by Invitrogen) was diluted 2500 times with TBS-B buffer and added to each well at 100 μL / well. After 1 hour and a half reaction at 30 ° C., each well was washed with the washing buffer.
(4-6) TMB Peroxidase Substrate (manufactured by KPL) was added and the absorbance at 450 nm was measured.

  (5) FIG. 5 shows the results of erythropoietin detection by ELISA using EPObp-m5 in Example 13 (4). The vertical axis in FIG. 5 is the ELISA measurement value (absorbance at 450 nm), and the horizontal axis is the logarithm of the erythropoietin concentration. Measurements were made in triplicate for each concentration of erythropoietin, and FIG. 5 was created based on the average value and standard deviation of the absorbance. As shown in FIG. 5, the absorbance value increased with increasing erythropoietin concentration. This shows that erythropoietin can be detected by the ELISA method using EPObp-m5. In addition, as shown in FIG. 5, since a standard curve can be created by plotting absorbance against erythropoietin concentration, it is possible to measure the concentration of erythropoietin at an unknown concentration by ELISA using EPObp-m5. It was suggested.

  Thus, it can be seen that EPObp-m5 is useful for a technique utilizing erythropoietin binding.

  The erythropoietin-binding protein of the present invention can be efficiently produced, and is useful as a ligand for affinity chromatography for purifying pharmaceuticals, biochemical reagents and erythropoietin including diagnostic agents from its erythropoietin-binding property.

Claims (7)

The following erythropoietin-binding protein (a) or (b):
(A) In the amino acid sequence represented by SEQ ID NO: 1, the erythropoietin-binding protein comprising the amino acid sequence in which one or more of the following amino acid substitutions (1) to (5) are substituted (1) The 103rd position of SEQ ID NO: 1 Arginine is replaced with serine (2) 141st histidine of SEQ ID NO: 1 is replaced with tyrosine (3) 159th alanine of SEQ ID NO: 1 is replaced with aspartic acid (4) 189th alanine of SEQ ID NO: 1 is aspartic acid Or substituted with valine (5) the 208th cysteine of SEQ ID NO: 1 is substituted with tyrosine (b) in the amino acid sequence of the erythropoietin-binding protein of (a) above, one or more amino acids are deleted, substituted or added Erythropoietin-binding protein consisting of a specific amino acid sequence The erythropoietin-binding protein according to claim 1, comprising an amino acid sequence represented by any one of SEQ ID NO: 2 to SEQ ID NO: 10. A polynucleotide encoding the erythropoietin-binding protein according to claim 1. An expression vector comprising the polynucleotide according to claim 3. A transformant obtained by transforming a host with the expression vector according to claim 4. The transformant according to claim 5, wherein the host is Escherichia coli. An erythropoietin binding comprising two steps: a step of producing an erythropoietin-binding protein by culturing the transformant according to claim 5 or 6, and a step of recovering the erythropoietin-binding protein produced from the obtained culture. A method for producing sex protein.

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