JP2011223982A - T7rna polymerase-fused protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fused protein prepared by fusing T7RNA polymerase with a heterogeneous protein; to provide a method for purifying the fused protein; and to provide a method for producing the heterogeneous protein from the fused protein.SOLUTION: There is provided the T7RNA polymerase-fused protein prepared by fusing the heterogenous protein with the carboxy terminal of the T7RNA polymerase directly or through one or more residual groups of amino acids. The T7RNA polymerase-fused protein can simply be purified with a Cibacron Blue-immobilizing resin, and can further position-specifically be hydrolyzed with a protease to produce a desired heterogeneous protein.

Description

  The present invention relates to a T7 RNA polymerase fusion protein in which a heterologous protein is fused directly or via a linker consisting of one or more amino acids to the carboxyl terminus of T7 RNA polymerase, and a polynucleotide encoding the T7 RNA polymerase fusion protein. The present invention also includes a bacterium belonging to the genus Escherichia that has the polynucleotide and can express the polynucleotide to produce the T7 RNA polymerase fusion protein of the present invention, as well as the T7 RNA of the present invention using the bacterium of the genus Escherichia. The present invention relates to a method for producing a polymerase fusion protein. Furthermore, the present invention relates to a method for producing a heterologous protein, which comprises hydrolyzing the T7 RNA polymerase fusion protein of the present invention with a protease.

  In protein production using E. coli (Escherichia coli), when a foreign protein derived from an organism different from the host E. coli, such as a so-called heterologous protein, is produced, the produced heterologous protein is accumulated in the bacterial body. Inclusion bodies are often formed. Since the heterologous protein forming the inclusion body is biologically or biochemically inactive due to its presence, further solubilization and regeneration operations are further required to obtain an active protein.

  In order to solve these problems, a method is known in which a protein expressed as a soluble protein in E. coli is fused with a heterologous protein. Proteins to be fused to heterologous proteins include Solubleity enhancement tag (SET) consisting of C-terminal site of 10B sequence of T7 phage, GB1 domain, ubiquitin, Small ubiquitin-modified modifier (SUMO), thioredoxin (TRX), glutathione (TRX), Fusion expression using transferase (GST), maltose binding protein (MBP), N-utilization maintenance A (Nus A) (Non-patent Document 1) and fusion expression using chaperonin lacking the carboxyl terminus (Patent Document 1) Are known.

  In the case where T7 RNA polymerase, which is RNA polymerase derived from bacteriophage T7 phage, is fused to a heterologous protein and expressed, for the purpose of functioning T7 RNA polymerase in yeast, the yeast GAL4 protein is converted to the amino terminus of T7 RNA polymerase. An example in which expression is made to be fused with is known (Non-patent Document 2). It is known that the GAL4 protein itself is expressed as a protein having a function also in E. coli cells.

  On the other hand, there has been no example of expressing a vertebrate-derived polypeptide or a fusion protein in which a vertebrate-derived polypeptide is linked to the carboxyl terminus of T7 RNA polymerase as a soluble protein.

  In addition, a polypeptide obtained by substituting and / or deleting and / or adding one or more amino acids of a vertebrate-derived polypeptide or a vertebrate-derived polypeptide to the carboxyl terminus of T7 RNA polymerase There is no example of expressing a fusion protein linked to as a soluble protein.

  In addition, it is known that a resin to which Cibacron Blue is immobilized can be used for purification of T7 RNA polymerase (Non-patent Document 3), but there is no example used for purification of a protein fused with T7 RNA polymerase.

JP 2004-321141 A JP 2009-136153 A

Kato, et al., Biophysics, Vol. 48, 185-189 (2008) Ostrander E.M. A. Et al. Science, 249, 1261-1265 (1990) Das M.M. Et al., Preparative Biochemistry and Biotechnology, 28, 339-348 (1998).

  An object of the present invention is to provide a T7 RNA polymerase fusion protein useful as a fusion protein for expressing a heterologous protein as a soluble protein that does not require solubilization or regeneration, and purity of the T7 RNA polymerase fusion protein. It is an object of the present invention to provide a method for producing a heterogeneous protein with high purity by simply hydrolyzing the T7 RNA polymerase fusion protein in a site-specific manner.

  As a result of intensive studies to solve the above problems, the present inventors have found that a T7 RNA polymerase fusion protein in which a heterologous protein is linked to the carboxyl terminus of T7 RNA polymerase and possibly via a linker as a soluble protein in the cytoplasm of Escherichia coli. The inventors have found that the expression of the fusion protein and the nature of the fusion protein are advantageous when purifying the heterologous protein from the fusion protein and the fusion protein, and have completed the present invention.

  That is, the present invention relates to a T7 RNA polymerase fusion protein in which a heterologous protein is fused directly to the carboxyl terminus of T7 RNA polymerase represented by SEQ ID NO: 1 or via a linker consisting of one or more amino acids. A heterologous protein may refer to the full length of the protein or a polypeptide having a function that is part of the protein.

  The present invention also provides a T7 RNA polymerase fusion protein in which a vertebrate polypeptide is fused to the carboxyl terminus of T7 RNA polymerase represented by SEQ ID NO: 1 directly or via a linker consisting of one or more amino acids. Alternatively, one or more amino acids of a vertebrate polypeptide may be converted to another amino acid directly or via a linker consisting of one or more amino acids at the carboxyl terminus of T7 RNA polymerase shown in SEQ ID NO: 1. The present invention relates to a T7 RNA polymerase fusion protein in which a substituted and / or deleted and / or added polypeptide is fused.

  The present invention also relates to positions 1 to 225 of the human erythropoietin receptor shown in SEQ ID NO: 2 directly or via a linker consisting of one or more amino acids at the carboxyl terminus of T7 RNA polymerase shown in SEQ ID NO: 1. A polypeptide consisting of 1 to 166 of human erythropoietin shown in SEQ ID NO: 3, a polypeptide consisting of 1 to 274 of human FcγRI shown in SEQ ID NO: 4, or SEQ ID NO: 5 The present invention relates to a T7 RNA polymerase fusion protein in which a polypeptide consisting of positions 1 to 238 of the human growth hormone receptor is fused.

  The present invention also relates to positions 1 to 225 of the human erythropoietin receptor shown in SEQ ID NO: 2 directly or via a linker consisting of one or more amino acids at the carboxyl terminus of T7 RNA polymerase shown in SEQ ID NO: 1. A polypeptide consisting of 1 to 166 of human erythropoietin shown in SEQ ID NO: 3, a polypeptide consisting of 1 to 274 of human FcγRI shown in SEQ ID NO: 4, or SEQ ID NO: 5 T7 RNA polymerase fusion protein fused with a polypeptide in which one or more amino acids of the polypeptide consisting of positions 1 to 238 of human growth hormone receptor are substituted and / or deleted and / or added to other amino acids It is about.

  The present invention also relates to a polynucleotide encoding the T7 RNA polymerase fusion protein of the present invention, and a bacterium belonging to the genus Escherichia that expresses the polynucleotide and produces the fusion protein.

  The present invention also relates to a method for producing a T7 RNA polymerase fusion protein using the Escherichia bacterium of the present invention.

  Furthermore, the present invention relates to a method for producing a heterologous protein, which comprises hydrolyzing a T7 RNA polymerase fusion protein with one or more proteases.

  The present invention also relates to a method for producing a T7 RNA polymerase fusion protein, characterized by using Cibacron Blue immobilized resin.

  The present invention also relates to a method for producing a heterologous protein, characterized in that the heterologous protein produced by hydrolysis of the T7 RNA polymerase fusion protein of the present invention is separated using a Cibacron blue-immobilized resin. The present invention relates to a method for producing a heterologous protein, characterized in that a heterologous protein produced by hydrolyzing the T7 RNA polymerase fusion protein of the invention with a protease is separated using a Cibacron Blue-immobilized resin.

  When the T7 RNA polymerase fusion protein of the present invention is used, the target heterologous protein can be produced as a soluble fusion protein using, for example, Escherichia coli. The T7 RNA polymerase fusion protein of the present invention can also be purified using Cibacron Blue immobilized resin. In particular, the T7 RNA polymerase fusion protein of the present invention in which a heterologous protein of interest is linked to T7 RNA polymerase through a linker capable of site-specific hydrolysis by, for example, a protease, is excised from T7 RNA polymerase at a desired position, The target heterologous protein can be produced efficiently.

T7 RNA polymerase, human erythropoietin receptor (hEPOR), EPObpf40 of Examples 1, 2 and 3, EPObpf40H of Examples 4 and 5, EPObpf50 of Example 12, EPObpf50H of Examples 6, 7, 8 and 9, and Examples Schematic diagram of primary structure of 13 and 14 EPObpf60H Schematic diagram of the primary structure of T7 RNA polymerase, human erythropoietin receptor (hEPOR), EPObpf2H of Examples 1, 4 and 6, human erythropoietin (hEPO), EPOf2 of Example 16 Schematic diagram of the plasmid prepared in Example 1 The figure which shows the result of SDS-PAGE of EPObpf40 of Example 3 Schematic diagram of the plasmid prepared in Example 4 The figure which shows the result of SDS-PAGE of EPObpf40H of Example 5 Schematic diagram of the plasmid prepared in Example 6 The figure which shows the result of SDS-PAGE of EPObpf50H of Example 7 The figure which shows the result of SDS-PAGE of EPObpH of Example 10 The figure which shows the result of the Western blotting of EPObpH of Example 10 Schematic diagram of the plasmid prepared in Example 12 Schematic diagram of the plasmid prepared in Example 13 The figure which shows the result of SDS-PAGE of EPObpf60H of Example 14 Schematic diagram of the primary structure of T7 RNA polymerase, human erythropoietin (hEPO), EPOf60 of Examples 16, 17 and 18. Schematic diagram of the plasmid prepared in Example 16 The figure which shows the result of SDS-PAGE of EPOf60 of Example 17 The figure which shows the result of the western blotting of EPOf60 of Example 18. Schematic diagram of the primary structure of T7 RNA polymerase, FcγRI and FcBPf51H of Example 19 Schematic diagram of the plasmid prepared in Example 19 The figure which shows the result of SDS-PAGE of FcBPf51H of Example 20 The figure which shows the result of the western blotting of FcBPf51H hydrolyzed using the enterokinase of Example 21 The figure which shows the result of the enzyme immunoassay of FcBPf51H hydrolyzed using the enterokinase of Example 21 Schematic diagram of primary structure of T7 RNA polymerase, hGHR and hGHbpf51H of Example 22 Schematic diagram of the plasmid prepared in Example 22 The figure which shows the result of the enzyme immunoassay of hGHbpf51H of Example 23.

  The heterologous protein fused directly with the T7 RNA polymerase shown in SEQ ID NO: 1 according to the present invention or via a linker consisting of one or more amino acids is derived from an organism heterologous to Escherichia coli. Examples include foreign proteins, and specific examples include polypeptides derived from vertebrates such as mammals, birds, reptiles, amphibians, and fish.

  More specifically, the heterologous proteins include growth hormone, erythropoietin, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5. (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), subunit p35 of interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), granulocyte colony stimulating factor (G-CSF) Granulocyte-macrophage colony-stimulating factor (GM-CSF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 ( T-1), leukocyte inhibitory factor (LIF), oncostatin M (OSM), interferon α (IFNα), interferon β (IFNβ), interferon γ (IFNγ), tumor necrosis factor α (TNFα), tumor necrosis factor β ( Examples thereof include polypeptides derived from vertebrates such as TNFβ), leptin, prolactin, and the like, or the extracellular domain of each receptor of these polypeptides.

  In addition, FcγRI (CD64), FcγRIIa (CD32A), FcγRIIb (CD32B), FcγRIIIa (CD16A), FcγRIIIb (CD16B), FcεRI, FcεRII (CD23), FcαRI (CD89), Fcα / μR, FcRn, polymeric Ig receptor, etc. Examples include vertebrate polypeptides such as the extracellular domain of antibody receptors, the extracellular domain of cell adhesion molecules (cadherin, integrin, selectin, etc.), and the H and L chains of antibodies. Moreover, polypeptides derived from vertebrates such as enzymes, polypeptides that are components of oligomeric enzymes, enzyme-inhibiting proteins, and soluble domains of membrane-bound enzymes can be exemplified.

In addition, the heterologous protein used in the present invention has, for example, one or more mutations selected from the following (a) to (c) in addition to the vertebrate-derived polypeptide as described above. In addition, it may have the activity of the heterologous protein, such as enzyme activity or binding activity.
(A) A deletion of one or more amino acids in the sequence of amino acid residues constituting the polypeptide (a) One or more amino acids in the sequence of amino acid residues constituting the polypeptide Substituted with other amino acids (c) A sequence of amino acid residues constituting the above polypeptide with one or more amino acids added.

  An additional sequence such as polyhistidine, S-peptide, or c-myc peptide may be linked to the carboxyl terminus of the heterologous protein.

  A polynucleotide encoding a heterologous protein can be prepared by a known method. For example, for a polynucleotide encoding a target heterologous protein, a primer set comprising oligonucleotides is prepared using known information, and a commercially available cDNA or cDNA library containing a polynucleotide encoding the target heterologous protein, Alternatively, it may be prepared by performing polymerase chain reaction (PCR) using cDNA prepared by a known method as a template and amplifying the target polynucleotide.

  For the amplification reaction of the polynucleotide by PCR, for example, PrimeSTAR HS DNA Polymerase (trade name, manufactured by Takara Bio Inc.) can be used, and if conditions for amplifying the target polynucleotide are experimentally determined according to the attached protocol, Good. For example, in the reaction solution using the attached buffer, for example, 0.01 U / μL PrimeSTAR HS DNA Polymerase, 0.2 mM dNTP, the concentration of the primer used in the reaction is 0.3 μM at maximum, the plasmid DNA 0. The reaction may be examined at 2 to 20 pg / mL. The reaction temperature and time are, for example, 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, then 72 ° C. The number of seconds obtained by multiplying the number of DNA base pairs to be amplified by 0.06 is one cycle. The reaction may be performed from 25 cycles to 30 cycles. Further, the temperature at 55 ° C. for 5 seconds in the reaction cycle may be set to any temperature between 56 ° C. and 72 ° C. to experimentally determine the temperature at which DNA to be amplified is obtained.

  The polynucleotide amplification reaction by PCR may be performed based on a known method, and is not limited to the above-described method.

  Amplification by PCR using a primer set designed to substitute, delete and / or add amino acids using the polynucleotide prepared by the above preparation method as a template, or by error-prone PCR By performing amplification, a polynucleotide encoding a heterologous protein having one or more amino acid substitutions and / or deletions and / or additions can be prepared.

  The target DNA amplified by PCR is subjected to, for example, agarose electrophoresis of the PCR reaction solution containing the DNA, and then the gel is stained with ethidium bromide and irradiated with ultraviolet light having a wavelength of 302 nm or 312 nm, for example. This DNA is detected, the gel containing the target DNA is cut out, and the DNA is purified therefrom. The DNA can be purified using a commercially available kit. For example, MERmaid Kit (trade name, Qbiogene) or DNA Purification Kit, 15, UltraClean (trade name, MO Bio Laboratories) What is necessary is just to use according to length. The DNA detection method and purification method are not limited to these.

  The polynucleotide used for genetically producing the T7 RNA polymerase fusion protein of the present invention may be any polynucleotide encoding the T7 RNA polymerase fusion protein of the present invention, and those skilled in the art can prepare it by known methods. Can do.

  An example of a method for preparing a polynucleotide is a method using PCR. For example, a polynucleotide can be obtained by preparing T7 phage DNA from T7 phage, using it as a template, and performing PCR with a forward primer and a reverse primer made of appropriate oligonucleotides. For example, a desired polynucleotide can be obtained by preparing T7 phage DNA from T7 phage, using it as a template, and performing PCR with a forward primer and a reverse primer made of appropriate oligonucleotides. The forward primer and reverse primer used for PCR are, for example, GenBank accession No. What is necessary is just to produce with reference to the base sequence of T7 RNA polymerase registered into NP_041960.

  Next, for example, a polynucleotide encoding the T7 RNA polymerase fusion protein of the present invention can be prepared by performing PCR in which the following (A) to (F) are reacted in one reaction solution.

  Namely, a polynucleotide comprising a base sequence encoding a T7 RNA polymerase obtained by PCR amplification and a double-stranded polynucleotide (A) comprising a complementary strand thereof, and a heterologous protein to be ligated And a double-stranded polynucleotide (B) consisting of a complementary strand thereof, a forward primer (C) of an oligonucleotide encoding several amino acids from methionine at position 1 of T7 RNA polymerase, amino acids from alanine at position 883 to several residues upstream of T7 RNA polymerase And, for example, an oligonucleotide reverse primer (D) 5 ′ end complementary to an oligonucleotide that encodes a portion spanning several amino acids from the amino terminus of the heterologous protein to be ligated to several amino acids downstream The side is complementary to the 5 'end of (D) An oligonucleotide encoding the amino acid of the sequence following the amino terminal portion of the heterologous protein encoded by the sequence, that is, the oligo encoding the portion straddling both of the complementary strand side of (D) and (B) A nucleotide forward primer (E) and an oligonucleotide reverse primer (F) complementary to an oligonucleotide encoding several amino acids on the carboxyl terminal side including the carboxyl terminal of a heterologous protein to be ligated with T7 RNA polymerase ) In a single reaction solution, a polynucleotide encoding the T7 RNA polymerase fusion protein of the present invention can be prepared.

  A polynucleotide encoding the T7 RNA polymerase fusion protein of the present invention in which a heterologous protein is fused to the carboxyl terminus of T7 RNA polymerase via a linker consisting of one or more amino acids is used instead of the primer (D) described above. Prepared by using an oligonucleotide reverse primer (G) complementary to a polynucleotide encoding one or more amino acids between the carboxyl terminus derived from T7 RNA polymerase and the polynucleotide encoding the amino terminus of the heterologous protein. be able to.

  The polynucleotide encoding the T7 RNA polymerase fusion protein of the present invention having an additional sequence at the carboxyl terminus of the heterologous protein to be ligated to T7 RNA polymerase is the carboxyl of the heterologous protein to be fused instead of the primer (F) described above. It can be prepared by using an oligonucleotide (H) that is complementary to an oligonucleotide that encodes several amino acids on the carboxyl terminal side including the terminal and a sequence to be added to the carboxyl terminal.

  In addition, for example, by performing PCR in which (A), (B), (C), (E), (G), and (H) are reacted in one reaction solution, the carboxyl terminus of T7 RNA polymerase has 1 A polynucleotide encoding the T7 RNA polymerase fusion protein of the present invention in which a heterologous protein having an additional sequence at the carboxyl terminus is fused can be prepared through a linker comprising amino acids of residues or more.

  For preparing a polynucleotide encoding the T7 RNA polymerase fusion protein of the present invention, for example, plasmids pTrc99A-T7RP and pCDF2-T7RP encoding T7 RNA polymerase (GenBank accession No. NP_041960) described in Patent Document 2 are prepared. It may be used as a template for PCR for preparing a polynucleotide encoding the T7 RNA polymerase fusion protein, and may further be used for the preparation of a plasmid encoding the polynucleotide encoding the T7 RNA polymerase fusion protein of the present invention. .

  In some cases, a linker comprising one or more amino acids may be inserted between the T7 RNA polymerase and the heterologous protein. The “linker consisting of one or more amino acids” may be any linker that can express the T7 RNA polymerase fusion protein of the present invention in a soluble manner, preferably a linker consisting of 1 to 50 amino acids, more preferably 1 Refers to a linker consisting of ˜20 amino acids. More preferably, it refers to a linker consisting of 1 to 10 amino acids, or a linker consisting of one or several amino acids.

  As the linker, for example, a linker having an amino acid sequence including an Asp-Pro sequence and / or an Asn-Gly sequence may be used.

  It is known that the Asp-Pro sequence is hydrolyzed at the bond between aspartic acid (Asp) and proline (Pro) with, for example, a 70% aqueous formic acid solution (pH 2.5). The Asn-Gly sequence is known to hydrolyze the bond between asparagine (Asn) and glycine (Gly) with, for example, a 2M hydroxylamine aqueous solution (pH 9).

  When the Asp-Pro sequence or Asn-Gly sequence is present in the heterologous protein molecule, the amino acid at that position may be substituted in advance. Aspartic acid (Asp) of the Asp-Pro sequence in the heterologous protein molecule may be substituted with an amino acid, for example, glutamic acid (Glu). Further, asparagine (Asn) in the Asn-Gly sequence in the molecule of the heterologous protein may be substituted with an amino acid, for example, glutamine (Gln) or, for example, aspartic acid (Asp).

  The linker used in the present invention may be, for example, a peptide consisting of an amino acid sequence that serves as a substrate for one or more types of protease (hereinafter referred to as substrate amino acid sequence). In particular, a protease having a high hydrolytic position specificity recognizes a specific amino acid sequence as a substrate and can selectively hydrolyze a specific peptide bond. When the T7 RNA polymerase fusion protein of the present invention having the above is hydrolyzed with a corresponding protease, the T7 RNA polymerase and the heterologous protein can be separated at a desired position.

  For example, enterokinase has four consecutive aspartic acid residues (Asp) followed by a lysine residue (Lys) as a protease having high position specificity of hydrolysis and its substrate amino acid sequence. -Asp-Lys amino acid sequence, thrombin is leucine residue-valine residue-proline residue-arginine residue (Lue-Val-Pro-Arg) amino acid sequence, activated blood coagulation factor X is isoleucine residue -Glutamic acid residue-Glycine residue-Arginine residue (Ile-Glu-Gly-Arg) amino acid sequence, Urokinase is proline residue-Glycine residue-Arginine residue (Pro-Gly-Arg) amino acid sequence Amino that is recognized as a substrate and bound to the carboxyl terminus of each substrate amino acid sequence Selectively hydrolyzing the peptide bond between. Therefore, when the amino terminus of a heterologous protein is bound to the carboxyl terminus of these substrate amino acid sequences, the heterologous protein can be easily excised by hydrolysis with the corresponding protease.

  In addition, for example, HRV 3C protease is leucine residue-glutamic acid residue-valine residue-leucine residue-phenylalanine residue-glutamine residue-glycine residue-proline residue (Leu-Glu-Val-Leu-Phe- A substrate amino acid sequence consisting of (Gln-Gly-Pro) is recognized, and the peptide bond between Gln and Gly of the sequence is hydrolyzed in a position-specific manner. Therefore, for example, when the amino terminus of a heterologous protein is bound to the carboxyl terminus of the substrate amino acid sequence consisting of Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro, it is hydrolyzed with HRV 3C protease. A heterologous protein having Gly-Pro added to the amino terminus can be obtained, and the present invention encompasses these heterologous proteins.

  A combination of a protease having high position specificity of hydrolysis and its substrate amino acid sequence is not particularly limited thereto.

  Furthermore, as a linker used in the present invention, a linker comprising an amino acid sequence in which one or more amino acids are added to the amino terminal side and / or the carboxyl terminal side of the substrate amino acid sequence of the above-mentioned protease having high positional specificity. In the case of using a linker consisting of an amino acid sequence in which one or more amino acids are added to at least the carboxyl terminal side of the substrate amino acid sequence, at least one residue or more is obtained by hydrolysis with a corresponding protease. The heterologous protein with the amino acid added to the amino terminus is obtained, but the present invention encompasses these heterologous proteins.

  In addition, when using a linker consisting of amino acid sequences that serve as substrates for multiple types of proteases, the substrate amino acid sequence closest to the amino terminus of the heterologous protein may be hydrolyzed with the corresponding protease, and as a result, one or more residues Even when a heterologous protein having the amino acid added at the amino terminus is obtained, the present invention encompasses these heterologous proteins.

  If proline is present at the carboxyl terminal side of the substrate amino acid sequence, hydrolysis may not occur. It is desirable to use the properties of the protease used and the substrate sequence after examining them in known literature.

  In hydrolysis using a protease, the reaction conditions and the like are determined so that the protease to be used, the T7 RNA polymerase fusion protein of the present invention, and the heterologous protein produced by hydrolysis of the protease do not insolubilize. That's fine. For example, the reaction may be performed under conditions appropriately selected from pH 5 to 8 and reaction temperature appropriately selected from 4 ° C. to 40 ° C., and the present invention is not limited thereto. For example, when using an aspartic protease, a serine protease, a metalloprotease, a thiol protease, or a protease classified into those proteases and having heat resistance, a reaction condition corresponding to the protease may be searched for and used.

  Regarding the addition of additives to the reaction solution, the type of salt (for example, sodium salt, potassium salt, magnesium salt or calcium salt) and its concentration (for example, 0 to 1 M), the type of surfactant (for example, Tween 20 ( Trade name), Tween 80 (trade name), Triton X-100 (trade name), n-octyl-β-D-glucoside, n-octyl-β-D-mannoside, n-decyl-β-D-mannoside, etc.) And the concentration (for example, 0% to 1%), the type of reducing agent (for example, dithiothreitol, mercaptoethanol, glutathione, etc.) and the concentration (for example, 0 mM to 10 mM), the type of denaturing agent (for example, urea, guanidine hydrochloride, etc.) Protea using its concentration (0M to 2M), etc., or combinations thereof for regiospecific hydrolysis Ze, the protease may be determined to respond to T7RNA polymerase fusion protein and a heterologous protein occurring it is regiospecific hydrolysis of the present invention without causing insolubilization.

  More specifically, for example, when enterokinase is used as a highly specific protease, the T7 RNA polymerase fusion protein of the present invention having a linker comprising the amino acid sequence of Asp-Asp-Asp-Asp-Lys (ie, The T7 RNA polymerase fusion protein of the present invention, which is a substrate for enterokinase, may be reacted in a solution of Tris-HCl buffer (pH 8.0) at a concentration of 0.02 M to 0.1 M, for example. The solution for the reaction may be 0.001M to 0.02M calcium chloride and / or sodium chloride having a concentration of 1M or less, more preferably sodium chloride having a concentration of 0.3M or less, and / or a surfactant. For example, Tween 20 (trade name) can be added at a concentration of 0.01% to 0.2%.

  The reaction temperature is appropriately selected from 4 ° C. to 40 ° C., the reaction time is appropriately selected from several minutes to several days, and the protein concentration and entero of the T7 RNA polymerase fusion protein of the present invention as a substrate for enterokinase are determined. The ratio of the protein concentration of the kinase may be, for example, 1:10 to 1: 10000.

  Further, for example, an affinity ligand such as polyhistidine having affinity for nickel chelate resin, S-peptide having affinity for S-protein, or c-myc peptide having affinity for c-myc antibody may be used as a linker.

  The linker that links the T7 RNA polymerase and the heterologous protein may be used in combination of a plurality of types of linkers as described above.

  The polynucleotide encoding the T7 RNA polymerase fusion protein of the present invention can be expressed in various hosts using a corresponding plasmid used in the field of normal genetic engineering, but is easy to handle and easy to culture. In particular, bacteria having a host / vector system for genetic manipulation, especially Escherichia coli which is a genus of Escherichia are preferable.

  Plasmids for expressing the T7 RNA polymerase fusion protein of the present invention include, for example, pTrc99a (GenBank Accession No. U13872), pSTV28 (trade name, manufactured by Takara Bio Inc.), pCDF-1b, pRSF-1b (above, Examples include plasmids such as trade names, manufactured by Merck Ltd.).

  Examples of E. coli transformed with a plasmid for expressing the T7 RNA polymerase fusion protein of the present invention include E. coli. coli JM109, E. coli. coli HB101, E. coli. Although strains such as E. coli BLR (DE3) can be exemplified, the present invention is not limited to these. In addition, when the E. coli before transformation with the plasmid for expressing the T7 RNA polymerase fusion protein of the present invention is E. coli having drug resistance, the drug resistance on the plasmid for expressing the T7 RNA polymerase fusion protein of the present invention. A marker gene different from the drug resistance of E. coli serving as a host may be selected. In the case of using a plasmid utilizing the T7 promoter for the expression of the T7 RNA polymerase fusion protein, for example, E. coli having a T7 RNA polymerase gene such as E. coli BLR (DE3) and expressing it may be used as a host.

  The transformed E. coli may be selected by culturing in an agar medium to which a drug corresponding to the drug resistance marker gene encoded in the plasmid is added.

  E. coli transformed with an expression plasmid incorporating a polynucleotide encoding the T7 RNA polymerase fusion protein of the present invention is fused with a T7 RNA polymerase fusion to a known medium for culturing E. coli used as a host, such as an LB medium or 2xYT medium. A drug corresponding to a drug marker gene on an expression plasmid into which a polynucleotide encoding a protein is incorporated may be added and cultured. The culture temperature may be determined experimentally at 15 to 37 ° C. In order to express the T7 RNA polymerase fusion protein of the present invention as a soluble protein in Escherichia coli transformed with an expression plasmid incorporating a polynucleotide encoding the T7 RNA polymerase fusion protein of the present invention, the protein is appropriately selected from 15 ° C to 37 ° C. The culture may be performed at a selected temperature, preferably a temperature appropriately selected from 25 ° C to 30 ° C.

  The T7 RNA polymerase fusion protein of the present invention can be obtained by appropriately culturing and proliferating E. coli transformed with an expression plasmid incorporating a polynucleotide encoding the same, and adding an appropriate inducer to the medium as necessary. After further culturing while inducing expression, the bacteria can be collected and disrupted by freeze-thawing, lysozyme digestion, sonication, or a combination thereof to obtain a soluble fraction.

  The T7 RNA polymerase fusion protein of the present invention contained in the soluble fraction can be purified using ion exchange chromatography or gel filtration chromatography.

  In addition, for example, a T7 RNA polymerase fusion protein to which an S-peptide tag is added can be purified using a carrier on which S-protein is immobilized, for example, S-protein agarose (trade name, manufactured by Merck).

  In the case of a T7 RNA polymerase fusion protein to which a so-called histidine tag made of polyhistidine is added, a resin chelated with a metal ion such as nickel ion or cobalt ion, for example, His · Bind Resin (trade name, manufactured by Merck & Co., Ltd.), Chelating Sepharose It can be purified using Fast Flow (trade name, manufactured by GE Healthcare Bioscience).

  Further, the T7 RNA polymerase fusion protein of the present invention can be adsorbed to a resin (referred to as Cibacron Blue-immobilized resin) immobilized with Cibacron Blue as a ligand. Cibacron Blue includes Cibacron Blue F3G-A [12236-82-7] and Cibacron Blue 3G-A [84116-13-2].

  Since the adsorption of the T7 RNA polymerase fusion protein of the present invention to Cibacron Blue-immobilized resin is hindered in the presence of the polynucleotide, when the polynucleotide is mixed in the solution of the T7 RNA polymerase fusion protein of the present invention in advance, It is desirable to leave out the polynucleotide.

  For example, the polynucleotide of the T7 RNA polymerase fusion protein solution of the present invention containing the polynucleotide can be precipitated by adding polyethyleneimine in the presence of salts. The salts added at this time may be any as long as they can prevent adsorption of the T7 RNA polymerase fusion protein to polyethyleneimine and coprecipitation with polyethyleneimine. Examples of salts to be added include sodium chloride, potassium chloride, and ammonium sulfate, and the concentration used and the concentration of polyethyleneimine to be used may be determined by experimenting conditions under which the T7 RNA polymerase fusion protein of the present invention is not insolubilized. For example, the salt concentration may be 0.1M to 0.5M, and the polyethyleneimine concentration may be 0.1% to 1%.

For example, E. coli disruption solution containing T7 RNA polymerase fusion protein, D. _{When 600} is a disrupted solution prepared from about 36 bacterial suspensions, polyethyleneimine is added to a concentration of 1 mg / mL in the presence of 0.2 M ammonium sulfate, and incubated for 30 to 60 minutes under ice cooling. After that, the precipitate can be removed by centrifugation.

  The method of removing nucleic acid is not limited to this, and any known method may be used.

  As Cibacron Blue-immobilized resin that adsorbs the T7 RNA polymerase fusion protein of the present invention, TOYOPEARL AF-Blue HC-650M (trade name, Tosoh Corporation) and Blue Sepharose 6 Fast Flow (trade name, GE Healthcare) are available. It can be illustrated.

  When the T7 RNA polymerase fusion protein of the present invention has a sequence capable of position-specific hydrolysis between T7 RNA polymerase and a heterologous protein, for example, a substrate amino sequence of a protease, it is hydrolyzed with the corresponding protease. Further, purification may be performed using Cibacron Blue fixing resin. Since the polypeptide containing T7 RNA polymerase is adsorbed on Cibacron Blue-immobilized resin, heterologous proteins that are not adsorbed on this resin can be separated from polypeptides containing T7 RNA polymerase and undigested fusion proteins.

  For example, in the case of the fusion protein of the present invention having an Asp-Asp-Asp-Asp-Lys sequence as a linker, which is an amino acid sequence that serves as a substrate for enterokinase, the fusion protein is converted to the carboxyl terminal side using enterokinase. After hydrolysis to T7 RNA polymerase having an Asp-Asp-Asp-Asp-Lys sequence and a heterologous protein, purification is performed using, for example, TOYOPEARL AF-Blue HC-650M (trade name, Tosoh Corporation). Just do it. At this time, the polypeptide containing T7 RNA polymerase is adsorbed on TOYOPEARL AF-Blue HC-650M. Even when a heterologous protein is adsorbed to the resin, elution is performed with a salt concentration gradient from 0 M to 2 M sodium chloride to separate it from T7 RNA polymerase having Asp-Asp-Asp-Asp-Lys sequence on the carboxyl terminal side. If it can be separated, it can be used for purification.

  Further, for example, the fusion protein of the present invention having an Asp-Asp-Asp-Asp-Lys sequence as a linker and a polyhistidine tag added to the carboxyl terminus of a heterologous protein is hydrolyzed using enterokinase, By purifying with a Clonblue immobilization resin and further purifying with, for example, a nickel chelate resin, a heterologous protein having a polyhistidine tag added to the carboxyl terminus can be purified.

  If necessary, the used protease may be removed using a resin in which a protease inhibitor or antibody that binds to the protease used for hydrolysis of the linker is immobilized.

Further, if there is a resin on which an antibody against an fused heterologous protein or an affinity ligand is immobilized, the heterologous protein may be purified using that resin.
Further, the above purification methods may be combined.

  In order to describe the present invention in more detail below, examples will be shown, but these examples show examples of the present invention, and the present invention is not limited to these examples.

Example 1 Construction of Plasmid pCDF20-EPObpf40 A fusion protein (EPObpf40, SEQ ID NO: 1) in which a polypeptide consisting of positions 1 to 225 of the human erythropoietin receptor (SEQ ID NO: 2) is linked to the carboxyl terminus of T7 RNA polymerase (SEQ ID NO: 1). 7, plasmid pCDF20-EPObpf40 (FIG. 3) encoding the polynucleotide (SEQ ID NO: 8) encoding (see FIG. 1).

  First, a 5.9 kbp linear DNA obtained by digesting the plasmid pCDF2-T7RP described in Patent Document 2 with the restriction enzyme XbaI was used to smooth the ends of the DNA using DNA Blunting Kit (trade name, manufactured by Takara Bio Inc.). Then, ligation was performed using DNA ligase and cyclization was performed to prepare a 5.9 kbp plasmid pCDF20-T7RP that does not contain the restriction enzyme XbaI site. PCR was performed using the plasmid as a template and the primers of SEQ ID NO: 11 and SEQ ID NO: 12 as a primer set to obtain 0.1 kbp DNA (referred to as DNA-1).

  The linear DNA obtained by digesting plasmid pCDF20-T7RP with restriction enzyme BlnI and then dephosphorylating with bovine intestinal alkaline phosphatase was further digested with restriction enzyme MfeI to obtain 5.8 kbp DNA (DNA-2 Called). On the other hand, using a plasmid encoding human erythropoietin receptor cDNA (manufactured by OriGene, product number SC125440) as a template, PCR was performed using the primer set of SEQ ID NO: 13 and SEQ ID NO: 14 to synthesize 0.7 kbp DNA, A 0.7 kbp DNA (referred to as DNA-3) obtained by double digesting the DNA with restriction enzymes MfeI and XbaI was obtained.

  The obtained DNA-2 and DNA-3 were ligated using DNA ligase and cyclized to prepare plasmid pCDF20-EPObpf2H.

  Plasmid pCDF20-EPObpf2H is a human erythropoietin receptor 1 shown in SEQ ID NO: 2 through the amino acid sequence from methionine 1 to 859 aspartate of T7 RNA polymerase, followed by 4 residues of Asp-Asp-Asp-Lys. This is a plasmid encoding a polynucleotide (SEQ ID NO: 10) encoding a fusion protein (EPObpf2H, SEQ ID NO: 9, see FIG. 2) in which a position from alanine to 225 aspartic acid followed by 6 histidines are linked.

  PCR was performed using the set of pCDF2DF20-EPObpf2H and DNA-1 (0.1 kbp) as a template and the primers of SEQ ID NO: 11 and SEQ ID NO: 13 as a primer set to obtain 0.8 kbp of DNA. The DNA was digested with restriction enzyme MfeI and then with restriction enzyme XbaI to obtain 0.8 kbp DNA (referred to as DNA-4).

  The DNA-4 was obtained by digesting the plasmid pCDF20-T7RP with the restriction enzyme BlnI and then dephosphorylating with bovine intestinal alkaline phosphatase, and further digesting the linear DNA with the restriction enzyme MfeI (5.8 kbp). Was ligated with the DNA (referred to as DNA-5) and cyclized to prepare a plasmid.

  E. coli HB101 was transformed with the plasmid, and a solid medium of 20 g / L agar (referred to as LB / Carb agar medium) containing 50 μg / mL carbenicillin sodium, 5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride. To obtain Escherichia coli HB101 / pCDF20-EPObpf40 having the target plasmid.

Example 2 Preparation of Fusion Protein EPObpf40 E. coli HB101 / pCDF20-EPObpf40 was added to a medium (referred to as 2xYT / Carb medium) containing 50 mg / L carbenicillin sodium, 10 g / L yeast extract, 16 g / L tryptone, 5 g / L sodium chloride. After inoculation and culturing at 25 ° C. for 16 hours, 0.12 L of a culture solution having a microbial density (OD ₆₀₀ ) of 6.8 was obtained.

E. coli HB101 / pCDF20-EPObpf40 was collected from the culture by centrifugation, suspended in 0.05 M Tris-HCl buffer (pH 8.0) containing 0.001 M ethylenediaminetetraacetic acid (EDTA), After washing the bacteria by centrifugation, 0.1 mg / mL egg white lysozyme, 0.0001M 4- (aminoethyl) benzenesulfonylfluoride so that the value of the bacterial density (OD ₆₀₀ ) is about 36. The cells were suspended in 0.05 M Tris-HCl buffer (pH 8.0) containing 0.0001 M phenylmethanesulfonyl fluoride, 1% (V / V) dimethyl sulfoxide and 0.001 M EDTA. The suspension was incubated at 0 ° C. for 1 hour, then mixed with 1/500 volume of 0.5 M dithiothreitol and frozen at −80 ° C. After thawing it, it was subjected to an ultrasonic crusher, and the liquid obtained by crushing was centrifuged to obtain a soluble fraction.

Example 3 Purification of fusion protein EPObpf40 using Cibacron Blue Immobilization Resin The soluble fraction obtained in the same manner as in Example 2 was sterilized by filtration with a cartridge filter (pore size 0.2 μm). / 10 volume of 0.22M ammonium sulfate aqueous solution was mixed, and 50 mg / mL polyethyleneimine aqueous solution of 1/50 volume of the mixed solution was mixed. After incubation at 0 ° C. for 1 hour, the insoluble nucleic acid was removed by centrifugation to obtain a soluble fraction, which was dissolved in 0.02 M sodium phosphate buffer (pH 7.5) containing 0.3 M sodium chloride. ) (Referred to as equilibration buffer 1) and dialyzed overnight at 4 ° C. Centrifugation after dialysis gave a soluble fraction that was sterile filtered to give 22.7 mL of filtrate.

  20 mL of the solution was loaded onto a column packed with 4 mL of TOYOPEARL AF-Blue HC-650M resin equilibrated with equilibration buffer 1. The resin was washed 8 times with 8 mL of equilibration buffer 1 each time, and the adsorbed fraction was eluted with 0.02 M sodium phosphate buffer (pH 7.5) containing 2 M sodium chloride. Sorted in size 2 mL.

  Each of the obtained fractions was added to an equal volume of 20 mg / mL sodium dodecyl sulfate (SDS), 40 mg / mL 2-mercaptoethanol, 570 mg / mL glycerin and 1 mg / mL bromophenol blue 0.25 M Tris-HCl buffer ( After adding pH 6.8) (referred to as SDS sample buffer) and heating to 98 ° C. for 5 minutes to form SDS, analysis was performed by SDS-polyacrylamide electrophoresis (SDS-PAGE).

  FIG. 4 shows lanes 1 and 14 of molecular weight marker proteins (175 kDa, 80 kDa, 58 kDa, 46 kDa, 30 kDa, 25 kDa in order from the top), Escherichia coli HB101 / pCDF20-T7RP (see Example 1) lysate (lane 2; size comparison sample) ), Sample before fractionation with TOYOPEARL AF-Blue HC-650M resin (lane 3), fraction passed through resin (lane 4), fraction obtained by washing resin (lanes 5, 6 and 7), The fraction obtained by eluting the fraction adsorbed on the resin (lanes 8 to 13 in the order of elution) was electrophoresed, and then stained with Coomassie Brilliant Blue R-250 (referred to as CBB staining) to detect the protein. It is a result.

  It can be seen that EPObpf40 (molecular weight 124 kDa) indicated by the arrow head (black triangle) is detected in lanes 9 and 10 of FIG. 4 and adsorbed on the TOYOPEARL AF-Blue HC-650M resin.

  As a result of colorimetric determination of the protein concentration of the obtained fraction using a protein assay (trade name, Bio-Rad) using γ-globulin as a standard protein, the total amount of the fractioned sample was 159 mg (100%). A fraction 35 mg (22%) containing EPObpf40 was obtained by removing 87 mg (55%) of the impure protein in a combined amount of the fraction passed through the resin and the fraction obtained by washing the resin.

Example 4 Construction of Plasmid pCDF20-EPObpf40H A polypeptide consisting of positions 1 to 225 of the human erythropoietin receptor (SEQ ID NO: 2) was linked to the carboxyl terminus of T7 RNA polymerase (SEQ ID NO: 1), followed by 6 histidines. Plasmid pCDF20-EPObpf40H (FIG. 5) encoding a polynucleotide (SEQ ID NO: 16) comprising a sequence (EPObpf40H, SEQ ID NO: 15, see FIG. 1) encoding a fusion protein ligated to each other was prepared.

  PCR was performed using plasmid pCDF20-EPObpf2H and DNA-1 (0.1 kbp) of Example 1 as a template and primers of SEQ ID NO: 11 and SEQ ID NO: 17 as a primer set to obtain 0.8 kbp of DNA. . The DNA was digested with restriction enzyme MfeI and then with restriction enzyme BlnI to obtain 0.8 kbp DNA (referred to as DNA-6). Subsequently, DNA-6 was ligated with DNA-5 of Example 1 and cyclized to prepare a plasmid.

  E. coli HB101 was transformed with the plasmid and cultured in LB / Carb agar medium to obtain E. coli HB101 / pCDF20-EPObpf40H having the target plasmid.

Example 5 Purification of fusion protein EPObpf40H In the same manner as in Example 2, E. coli HB101 / pCDF20-EPObpf40H was cultured to obtain 0.12 L of a culture solution having a bacterial density (OD ₆₀₀ ) of 6.6. A soluble fraction was obtained from Escherichia coli obtained from the culture solution in the same manner as in Example 2.

  The soluble fraction (the fraction containing EPObpf40H) is sterilized by filtration with a cartridge filter (pore size 0.2 μm), mixed with 1/10 volume of 0.22 M aqueous ammonium sulfate solution, and then 1 / of the mixture. 49 volumes of 50 mg / mL aqueous polyethyleneimine solution were mixed. It was incubated at 0 ° C. for 1 hour and then centrifuged to obtain a soluble fraction. It was dialyzed overnight at 4 ° C. in 0.02 M sodium phosphate buffer (pH 7.5) containing 0.3 M sodium chloride, and then 1/19 volume of 1 M aqueous imidazole was added. Nickel chelate resin (His Bind Resin) equilibrated with 0.02M sodium phosphate buffer (pH 7.5) (referred to as equilibration buffer 2) containing 0.05M imidazole and 0.3M sodium chloride. (Trade name, manufactured by Merck & Co., Inc.) was loaded onto a column packed with 2 mL, and 5 mL of equilibration buffer 2 was loaded to wash the resin, and pooled as a flow-through fraction. The resin was further washed with 15 mL of equilibration buffer 2 and washed with 0.02 M sodium phosphate buffer (pH 7.5) containing 0.2 M imidazole and 0.3 M sodium chloride. The elution fraction was eluted and collected.

  In FIG. 6, lanes 1 and 11 are a molecular weight marker protein, a sample before fractionation with a nickel chelate resin (lane 2), a fraction passed through the resin (lane 3), and a fraction obtained by washing the resin (lane 4). ), The fraction obtained by eluting the fraction adsorbed on the resin (in the order of elution, lanes 5 to 10), after electrophoresis, the protein was detected by CBB staining. EPObpf40H was detected in lanes 6 to 9, indicating that it was purified compared to the sample in lane 2.

  The protein concentration of the fraction of EPObpf40H (molecular weight 124 kDa) adsorbed on the nickel chelate resin was colorimetrically determined by protein assay using γ-globulin as a standard protein.

  The EPObpf40H obtained in this example was 2.0 mg.

Example 6 Construction of Plasmid pCDF20-EPObpf50H 1 of human erythropoietin receptor (SEQ ID NO: 2) via 6 residues of Gly-Asp-Asp-Asp-Asp-Lys at the carboxyl terminus of T7 RNA polymerase (SEQ ID NO: 1) Encoded a polynucleotide (SEQ ID NO: 19) encoding a fusion protein (EPObpf50H, SEQ ID NO: 18, see FIG. 1) in which a polypeptide consisting of positions 225 to 225 was linked, followed by 6 histidines linked, Plasmid pCDF20-EPObpf50H (FIG. 7) was prepared.

  PCR was performed using the plasmid pCDF20-T7RP as a template and the primers of SEQ ID NO: 11 and SEQ ID NO: 20 as a primer set to obtain 0.1 kbp DNA (referred to as DNA-7).

  PCR was performed using plasmid pCDF20-EPObpf2H as a template and the primer of SEQ ID NO: 17 and the above DNA-7 as a primer set to obtain 0.8 kbp of DNA. The DNA was digested with restriction enzyme MfeI and then with restriction enzyme BlnI to obtain 0.8 kbp DNA (referred to as DNA-8).

  DNA-8 was ligated with DNA-5 of Example 1 and cyclized to prepare a plasmid.

  E. coli HB101 was transformed with the plasmid and cultured in LB / Carb agar medium to obtain E. coli HB101 / pCDF20-EPObpf50H having the target plasmid.

Example 7 Purification of fusion protein EPObpf50H In the same manner as in Example 2, E. coli HB101 / pCDF20-EPObpf50H was cultured to obtain 0.12 L of a culture solution having a bacterial density (OD ₆₀₀ ) of 6.6. A soluble fraction was obtained from Escherichia coli obtained from the culture solution in the same manner as in Example 2 except that dithiothreitol was not used.

  The soluble fraction (the fraction containing EPObpf50H) was purified using a nickel chelate resin in the same manner as in Example 5.

  Lanes 1 and 11 in FIG. 8 are a sample before fractionation with a molecular weight marker protein and nickel chelate resin (lane 2), a fraction passed through the resin (lane 3), and a fraction obtained by washing the resin (lane 4). The results of eluting the fraction adsorbed on the resin (in the order of elution, lanes 5 to 10) after electrophoresis were subjected to CBB staining and the protein was detected. The fractions electrophoresed in the indicated lanes 6 and 7 contained a large amount of EPObpf50H.

  The protein concentration of the fraction of EPObpf50H (molecular weight 125 kDa) adsorbed on the nickel chelate resin was colorimetrically determined by protein assay using γ-globulin as a standard protein.

  The EPObpf50H obtained in this example was 1 mg.

Example 8 Hydrolysis of fusion protein EPObpf50H by enterokinase The soluble fraction (fraction containing EPObpf50H) obtained in the same manner as in Example 7 was sterilized by filtration with a cartridge filter (pore size 0.2 μm), and then 36 ng of enterokinase (Enterokinase, light chain, trade name, NEW ENGLAND BioLabs) was added to 9 mL of the filtrate and incubated at 25 ° C. for 18 hours.

Example 9 Purification of EPObpH The fusion protein EPObpf50H was hydrolyzed with enterokinase, that is, it was hydrolyzed between positions 889 and 890 of SEQ ID NO: 18, and consisted of a sequence from positions 890 to 1120 of SEQ ID NO: 18. A polypeptide, ie, a polypeptide in which 6 residues of histidine were added to glutamic acid at position 1 to position 225 of human erythropoietin receptor (referred to as EPObpH) was purified as follows.

  1/10 volume of 0.22M ammonium sulfate aqueous solution was mixed with the soluble fraction incubated with the addition of enterokinase of Example 8, and then 1/50 volume of 50 mg / mL polyethyleneimine aqueous solution was mixed. After incubating it at 0 ° C. for 1 hour, it was centrifuged to remove insolubilized nucleic acid to obtain a soluble fraction, which was dialyzed overnight in equilibration buffer 1 at 4 ° C. After the dialysis, centrifugation was performed to obtain a soluble fraction.

  9 mL of the soluble fraction was loaded onto a column packed with 1.8 mL of TOYOPEARL AF-Blue HC-650M resin equilibrated with equilibration buffer 1, and a column non-adsorbed fraction was obtained. The total protein content of the fraction was 17.2 mg.

  After adding 1/19 volume of 1M imidazole aqueous solution to the non-adsorbed fraction, the column was loaded with 2 mL of nickel chelate resin (His · Bind Resin) equilibrated with equilibration buffer 2. The resin was washed by loading 10 mL of equilibration buffer 2 and the adsorbed fraction was eluted with 0.02 M sodium phosphate buffer (pH 7.5) containing 0.2 M imidazole and 0.3 M sodium chloride. The fraction was fractionated at 1 mL.

Example 10 Analysis of EPObpH After subjecting the fraction obtained in Example 9 to SDS-PAGE, 0.01M cyclohexylaminopropipansulfonic acid (CAPS) -sodium hydroxide containing 10% (V / V) methanol Electrophoresis was performed in a buffer solution (pH 11) (referred to as a CAPS buffer solution) and transferred to a PVDF membrane. The membrane was prepared with 5 mg / mL bovine serum albumin solution (BSA / T) prepared with 0.05 M Tris-HCl buffer (pH 7.5) (referred to as TBS-T) containing 0.15 M sodium chloride and 0.5 mg / mL Tween20. After blocking with a TBS-T solution), Western blotting was performed and detection was performed using an anti-human erythropoietin receptor antibody.

  The Western blotting procedure was as follows. First, the blocked membrane was immersed in a BSA / TBS-T solution containing 1 μg / mL mouse anti-human erythropoietin antibody, shaken at room temperature for 1 hour, washed with TBS-T, B / F separated. The membrane was then immersed in a BSA / TBS-T solution containing horseradish peroxidase (HRP) -labeled goat anti-mouse IgG (H + L) antibody at a concentration of 1 μg / mL and shaken at room temperature for 1 hour. After that, B / F was separated. Next, the HRP activity of the HRP-labeled goat anti-mouse IgG (H + L) antibody bound to the membrane is 24.3 mM citrate-51.4 mM disodium hydrogen phosphate containing 0.1% hydrogen peroxide and 5 mM 4-chloronaphthol. A color reaction was performed at room temperature for 20 minutes using a buffer solution (pH 5) as a substrate solution, and EPObpf50H and EPObpH were detected.

  FIG. 9 shows the results of SDS-PAGE, and FIG. 10 shows the results of Western blotting.

  Lanes 1 and 15 are molecular weight marker proteins, lane 2 is a soluble fraction before enterokinase treatment, lane 3 is a soluble fraction after enterokinase treatment, and TOYOPEARL AF-Blue HC-650M resin column non-adsorbed fraction (Lane 4), all fractions obtained by washing in the same manner as in Example 3 (lane 5), adsorbed fractions obtained in the same manner as in Example 3 (lanes 6 to 9 in the order of elution), nickel chelate The fraction loaded on the resin (lane 10; the same fraction as lane 4), the non-adsorbed fraction of the nickel chelate resin (lane 11) and the adsorbed fraction (lanes 12 to 14 in the order of elution), FIG. From the results of 10, it can be seen that there are purified EPObpH in lanes 13 and 14.

  As a result of colorimetric determination with a protein assay using γ-globulin as a standard protein, the obtained EPObpH was 0.29 mg.

Example 11 Adsorption of human erythropoietin to EPObpH-binding resin Escherichia coli HB101 / pCDF20-EPObpf50H was cultured to obtain Example 9 from bacteria obtained from 0.5 L of a culture solution having a cell density (OD ₆₀₀ ) of 5.3. In the same manner, an EPObpH fraction (total protein mass: 1 mg) was obtained. The TOPOEARL AF-Blue HC-650M resin was equilibrated with 0.02 M sodium phosphate buffer (pH 7.5) containing 0.2 M imidazole and 0.3 M sodium chloride. The column loaded with 2 mL was loaded, and 0.4 mg of EPObpH was obtained in the column non-adsorbed fraction.

  After adjusting the buffer composition of the EPObpH (0.24 mg) solution to 0.02 M sodium phosphate buffer (pH 7.5) containing 0.05 M imidazole and 0.3 M sodium chloride, the nickel chelate resin was added to a concentration of 0. Loaded on 1 mL packed column. The resin was washed with 20 mL of equilibration buffer 2. After 0.2 mL of CHO cell-derived recombinant human erythropoietin (manufactured by Merck) prepared to 0.25 mg / mL was loaded onto the resin, the resin was washed with 10 mL of the same composition buffer. Thereafter, elution was carried out with 5 mL of 0.02 M sodium phosphate buffer (pH 7.5) containing 8 M urea, 0.2 M imidazole and 0.3 M sodium chloride. The liquid flow through the column was performed at a flow rate of 0.1 mL / min.

  When the same operation was performed using a resin not bound with EPObpH, CHO cell-derived recombinant human erythropoietin was not adsorbed to the resin.

  Fractions (fraction size 1 mL) obtained using the EPObpH-bound resin were analyzed by enzyme immunoassay.

  In the enzyme immunoassay, 4 μg / mL rabbit anti-erythropoietin polyclonal antibody was added to a 96-well immunoplate, 100 μL of 0.05 M Tris-HCl buffer (pH 8.0) per well, and incubated at 4 ° C. for 18 hours. B / F separation was performed by rinsing with T, and then 200 μL of BSA / TBS-T was added per well, followed by incubation at 30 ° C. for 2 hours for blocking.

  The blocked plate was rinsed with TBS-T and subjected to B / F separation. A sample prepared by diluting with BSA / TBS-T was added at 100 μL per well and incubated at 30 ° C. for 1 hour. separated. Next, 0.8 μg / mL biotin-labeled anti-erythropoietin monoclonal antibody was added at 100 μL per well, incubated at 30 ° C. for 1 hour, B / F separation was performed, and 0.6 μg / mL HRP-labeled streptavidin was further added at 100 μL per well. In addition, it was incubated at 30 ° C. for 30 minutes.

  After B / F separation, 100 μL of TMB Microwell Peroxidase Substrate (2-Component System) (product name, Kirkegaard & Perry Laboratories) was added to each well, and reacted at 37 ° C. for 20 minutes. After stopping the reaction by adding 50 μL per well, the absorbance (OD450) at a wavelength of 450 nm was measured.

  As a result of analysis, about 35 μg of CHO cell-derived recombinant human erythropoietin was detected in the fraction passed through the resin bound with EPObpH, but 0.02M phosphoric acid containing 8M urea, 0.2M imidazole and 0.3M sodium chloride. About 15 μg was detected in the fraction eluted with sodium buffer (pH 7.5), and it was found that CHO cell-derived recombinant human erythropoietin was adsorbed to EPObpH on the resin.

Example 12 Construction of Plasmid pCDF20-EPObpf50 1 of human erythropoietin receptor (SEQ ID NO: 2) via 6 residues of Gly-Asp-Asp-Asp-Asp-Lys at the carboxyl terminus of T7 RNA polymerase (SEQ ID NO: 1) Plasmid pCDF20-EPObpf50 (FIG. 11) encoding a polynucleotide (SEQ ID NO: 22) encoding a fusion protein (EPObpf50, SEQ ID NO: 21, see FIG. 1) linking polypeptides from position 225 to position 225 was prepared. .

  Plasmid pCDF20-EPObpf50H was digested with restriction enzyme MfeI and then with EcoRV to obtain 0.4 kbp DNA (referred to as DNA-9).

  On the other hand, plasmid pCDF20-EPObpf40 was digested with restriction enzyme MfeI and then with EcoRV to obtain 6.2 kbp DNA (referred to as DNA-10).

  DNA-9 was ligated with the above DNA-10 and cyclized to prepare a plasmid.

  E. coli HB101 was transformed with the plasmid and cultured in LB / Carb agar medium to obtain E. coli HB101 / pCDF20-EPObpf50 having the target plasmid.

Example 13 Construction of Plasmid pCDF20-EPObpf60H From position 1 to 225 of human erythropoietin receptor (SEQ ID NO: 2) via the 5 residues of Gly-Ser-Pro-Gly-Arg at the carboxyl terminus of T7 RNA polymerase (SEQ ID NO: 1) Plasmid pCDF20− encoding a polynucleotide (SEQ ID NO: 24) encoding a fusion protein (EPObpf60H, SEQ ID NO: 23, see FIG. 1) in which a polypeptide consisting of up to 5 positions is linked, followed by 6 histidines linked EPObpf60H (FIG. 12) was produced.

  First, PCR was performed using the plasmid pCDF20-EPObp50H of Example 6 as a template and primers of SEQ ID NO: 17 and SEQ ID NO: 25 as a primer set to obtain 0.7 kbp DNA. The DNA was digested with the restriction enzyme BamHI to obtain 0.7 kbp DNA (referred to as DNA-11).

  On the other hand, PCR was performed using plasmid pCDF20-T7RP as a template and primers of SEQ ID NO: 11 and SEQ ID NO: 26 as a primer set to obtain 0.1 kbp DNA. The DNA was digested with the restriction enzyme BamHI to obtain 0.1 kbp DNA (referred to as DNA-12).

  After ligating DNA-11 and DNA-12, PCR was performed using the primers of SEQ ID NO: 11 and SEQ ID NO: 17 as a primer set to obtain 0.8 kbp of DNA. The DNA was digested with restriction enzyme MfeI and then with restriction enzyme BlnI to obtain 0.8 kbp DNA (DNA-13).

  DNA-13 was ligated with DNA-5 of Example 1 and cyclized to prepare a plasmid.

  Escherichia coli HB101 was transformed with the plasmid and cultured in LB / Carb agar medium to obtain Escherichia coli HB101 / pCDF20-EPObpf60H having the desired plasmid pCDF20-EPObpf60H.

Example 14 Expression of Fusion Protein EPObpf60H E. coli HB101 / pCDF20-EPObpf60H was inoculated into 2xYT / Carb medium and cultured overnight at 30 ° C. The culture solution was divided into two, IPTG was added to one of them to a concentration of 0.1 mM, and each was cultured at 25 ° C. for 2 hours.

The obtained bacterial cells were rinsed with 0.05 M Tris-HCl buffer (pH 8.0) containing 0.001 M EDTA, and O.D. D. _The sample was resuspended so that ₆₀₀ became 10, and the same volume of SDS sample buffer was added, heated at 98 ° C. for 5 minutes to form SDS, and then analyzed by SDS-PAGE.

  Lane 1 in FIG. 13 is a molecular weight marker protein, EPObpf50H (comparative sample, lane 2, without IPTG), EPObpf50H (comparative sample, lane 3, with IPTG), EPObpf60H (lane 4, without IPTG), EPObpf60H (with lane 5, IPTG) It can be seen that EPObpf60H (125 kDa) is expressed in lanes 4 and 5 regardless of whether IPTG is added or not.

Example 15 Purification of fusion protein EPObpf60H In the same manner as in Example 2, E. coli HB101 / pCDF20-EPObpf60H was cultured to obtain about 1 L of a culture solution having a bacterial density (OD ₆₀₀ ) of 4.8.
A soluble fraction was obtained from Escherichia coli (wet cell weight 4.6 g) obtained from the culture solution in the same manner as in Example 2.
The soluble fraction was dialyzed overnight at 4 ° C. in equilibration buffer 1, and imidazole was added to a final concentration of 0.05M.
The solution (EPObpf60H solution) was divided into two portions and purified as follows.
A column filled with 10 mL of nickel chelate resin (His Bind Resin, trade name, manufactured by Merck) equilibrated with equilibration buffer 2 (see Example 5) was loaded with a solution of EPObpf60H, and then 50 mL of equilibration was performed. After washing with buffer 2, the adsorbed fraction was eluted and fractionated with a 0.02 M sodium phosphate buffer (pH 7.5) containing 0.2 M imidazole and 0.3 M sodium chloride at a fraction size of 5 mL.
The protein concentration of the adsorbed fraction was colorimetrically determined by protein assay using γ-globulin as a standard protein. As a result, the total amount of EPObpf60H obtained was 68.8 mg.

Example 16 Construction of Plasmid pCDF20-EPOF60 A fusion protein in which erythropoietin (SEQ ID NO: 3) was linked to the carboxyl terminus of T7 RNA polymerase (SEQ ID NO: 1) via 5 residues of Gly-Ser-Pro-Gly-Arg Plasmid pCDF20-EPOf60 (FIG. 15) encoding a polynucleotide (SEQ ID NO: 28) encoding EPOf60, SEQ ID NO: 27, FIG. 14) was prepared.

  First, it amplified by PCR using the primer set of sequence number 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 and 48. A DNA (referred to as DNA-14) obtained by double digesting 0.34 kbp DNA with restriction enzymes MfeI and PstI was obtained.

  In addition, a 0.25 kbp DNA amplified by PCR using the primer sequences of primer sequence numbers 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60 was used as a restriction enzyme. DNA double-digested with PstI and BlnI (referred to as DNA-15) was obtained. DNA-5 described in Example 1, DNA-14 and DNA-15 were ligated using DNA ligase and cyclized to prepare plasmid pCDF20-EPOf2.

  The plasmid pCDF20-EPOf2 is shown in SEQ ID NO: 3 through the amino acid sequence from the 1st methionine to the 859th aspartic acid of T7 RNA polymerase (SEQ ID NO: 1), via the 4 residues of Asp-Asp-Asp-Lys. It is a plasmid encoding a polynucleotide (SEQ ID NO: 30) encoding a fusion protein (EPObpf2, SEQ ID NO: 29, see FIG. 2) in which human erythropoietin is linked from position 1 alanine to position 166 arginine.

  PCR was performed using the plasmid pCDF20-EPOf2 template and the primer set of SEQ ID NO: 60 and SEQ ID NO: 61 to obtain 0.5 kbp of DNA. The DNA was digested with the restriction enzyme BamHI and 0.5 kbp of DNA ( (Referred to as DNA-16).

  After ligating DNA-12 of Example 13 and DNA-16, PCR was performed using the primer sets of SEQ ID NO: 11 and SEQ ID NO: 60 to obtain 0.6 kbp DNA. The DNA was digested with the restriction enzyme MfeI and then with the restriction enzyme BlnI to obtain 0.6 kbp DNA (referred to as DNA-17).

  DNA-17 was ligated with DNA-5 of Example 1 and cyclized to prepare a plasmid.

  Escherichia coli HB101 was transformed with the plasmid and cultured in LB / Carb agar medium to obtain Escherichia coli HB101 / pCDF20-EPOf60 having the target plasmid.

Example 17 Expression of Fusion Protein EPOf60 E. coli HB101 / pCDF20-EPOf60 was inoculated into 2xYT / Carb medium and cultured overnight at 30 ° C. The culture solution was divided into two, and isopropyl-1-thio-β-D-galactoside (IPTG) was added to one of them to a concentration of 0.1 mM, and each was cultured at 25 ° C. for 2 hours.

After rinsing the obtained bacterial cells with 0.05 M Tris-HCl buffer (pH 8.0) containing 0.001 M EDTA, the bacterial density was set to O.D. D. _After resuspending at ₆₀₀ to 10 and adding the same volume of SDS sample buffer and heating at 98 ° C. for 5 minutes to form SDS, analysis was performed by SDS-PAGE.

  In FIG. 16, lane 1 is a molecular weight marker protein, lane 2 is EPOf60 (without IPTG), and lane 3 is EPOf60 (with IPTG). It was found that EPOf60 (118 kDa) was expressed at the position indicated by the arrow in lane 3.

Example 18 Hydrolysis of fusion protein EPOf60 by urokinase E. coli HB101 / pCDF20-EPOf60 D. _After inoculating in 2xYT / Carb medium to 0.2 at ₆₀₀ , culturing at 37 ° C for 3 hours, adding IPTG to a concentration of 0.1 mM, and further culturing at 25 ° C for 3 hours .

After rinsing the obtained cells with 0.05M Tris-HCl buffer (pH 8.0) containing 0.001M EDTA, 0.05M Tris-HCl buffer containing 0.1 mg / mL egg white lysozyme and 0.001M EDTA (PH 8.0) with O.D. D. _It was resuspended so that ₆₀₀ became 36, incubated for 1 hour under ice-cooling, and then frozen at -80 ° C. After thawing, it was sonicated and then centrifuged to obtain a supernatant. It was sterile filtered through a cartridge filter (pore size 0.2 μm), and the filtrate was used as a soluble fraction. To 200 μL of the soluble fraction, 0.3 mg / mL natural urokinase (enzyme that activated the natural prourokinase-like polypeptide described in JP-A-62-143686) or 0.3 mg / ml mutant urokinase (special 50 μL of the mutant (Q135D157) prourokinase-like polypeptide-activated mutant described in Kaisho 62-143686 was added and incubated at 4 ° C.

  A part of the solution incubated at 4 ° C. for 68 hours was subjected to SDS-PAGE, then electrophoresed in a CAPS buffer, and transferred to a PVDF membrane. After blocking the membrane in a BSA / TBS-T solution, Western blotting analysis was performed and detected using an anti-human erythropoietin antibody.

  The Western blotting procedure was as follows. First, the blocked membrane was immersed in a BSA / TBS-T solution containing 0.5 μg / mL biotin-labeled mouse anti-human erythropoietin antibody, shaken at room temperature for 1 hour, and then TBS-T. And B / F separation was performed. Next, the membrane was immersed in a BSA / TBS-T solution containing 0.6 μg / mL horseradish peroxidase (HRP) -labeled streptavidin, shaken at room temperature for 1 hour, and B / F separated. Next, the HRP activity of the HRP-labeled streptavidin bound to the membrane was determined using 24.3 mM citrate-51.4 mM disodium hydrogen phosphate buffer (pH 5) containing 0.1% hydrogen peroxide and 5 mM 4-chloronaphthol. As a substrate solution, a color reaction was performed at room temperature for 20 minutes to detect, and erythropoietin was detected.

  Lane 1 in FIG. 17 is a soluble fraction of E. coli HB101 / pCDF20-EPOf60 (no urokinase) incubated for 68 hours, lane 2 is a soluble fraction of E. coli HB101 / pCDF20-EPOf60 incubated with natural urokinase for 68 hours, lane 3 shows the results of analysis of the soluble fraction of Escherichia coli HB101 / pCDF20-EPOf60, which was incubated for 68 hours with the addition of mutant urokinase. Erythropoietin (18 kDa) was detected at the position indicated by the arrow.

Example 19 Construction of Plasmid pCDF20-FcBPf51H Antibody receptor (FcγRI) (SEQ ID NO: 1) via the 7 residues of Gly-Ser-Asp-Asp-Asp-Asp-Lys at the carboxyl terminus of T7 RNA polymerase (SEQ ID NO: 1) 4) A polynucleotide (SEQ ID NO: 63) encoding a fusion protein (FcBPf51H, SEQ ID NO: 62, FIG. 18) in which a polypeptide consisting of positions 1 to 274 is linked, followed by 6 histidines. The encoded plasmid pCDF20-FcBPf51H (FIG. 19) was generated.

  First, PCR in the first stage was performed using the plasmid pECFcR described in JP-A-2008-245580 as a template using the primer set of SEQ ID NO: 64 and SEQ ID NO: 65, and 0.8 kbp of DNA (DNA-18 and Obtained).

  Next, PCR in the second stage was performed using plasmid pECFcR as a template and a primer set of DNA-18 and SEQ ID NO: 66 to obtain 0.9 kbp DNA (referred to as DNA-19).

  Next, the third-stage PCR is performed using DNA-19 as a template and the primer set of SEQ ID NO: 64 and SEQ ID NO: 67 to obtain 0.9 kbp of DNA, which is digested with the restriction enzyme BamHI. Thus, 0.9 kbp DNA (referred to as DNA-20) was obtained.

  The DNA-20 was ligated with a 6.1 kbp DNA (referred to as DNA-21) obtained by digesting the plasmid pCDF20-EPObpf60H with restriction enzymes NaeI and BamHI, and cyclized to prepare a plasmid.

  E. coli JM109 was transformed with the plasmid and cultured in LB / Carb agar medium to obtain E. coli JM109 / pCDF20-FcBPf51H having the target plasmid.

Example 20 Preparation of FcBPf51H E. coli JM109 / pCDF20-FcBPf51H was inoculated into 2xYT / Carb medium and cultured at 28 ° C. for 15 hours, and a culture solution having a bacterial density (OD ₆₀₀ ) of 8.3, 0. 12L was obtained.

  E. coli JM109 / pCDF20-FcBPf51H is collected from the culture by centrifugation, suspended in 25 mL of 0.05 M Tris-HCl buffer (pH 8.0) containing 0.001 M EDTA, and then centrifuged. The bacteria were washed. Next, the bacteria were washed with 25 mL of 0.05 M Tris-HCl buffer (pH 8.0).

The bacteria are suspended in 0.2 mg / mL egg white lysozyme, 0.05 M Tris-HCl buffer (pH 8.0) so that the density of the bacteria (OD ₆₀₀ ) is about 72. did. The suspension was incubated at 0 ° C for 1 hour and then frozen at -80 ° C. After thawing it, it was subjected to an ultrasonic crusher, and the liquid obtained by crushing was centrifuged to obtain a soluble fraction.

  The obtained soluble fraction was sterilized by filtration with a cartridge filter (pore size 0.2 μm), and 12.4 mL of the filtrate was mixed with 1/59 volume of 3M imidazole (pH 6).

  Nickel chelate resin (His) equilibrated with 12.4 mL of the solution with 0.05 M Tris-HCl buffer (pH 8.0) (referred to as equilibration buffer 3) containing 0.05 M imidazole and 0.3 M sodium chloride. After loading onto a column packed with 4 mL of Bind Resin (trade name, manufactured by Merck), the resin was washed with 4 mL of equilibration buffer 3 and pooled as a flow-through fraction. The resin was further washed with 16 mL of equilibration buffer 3, and then the adsorbed fraction was extracted with 0.05 M Tris-HCl buffer (pH 8.0) containing 0.2 M imidazole and 0.3 M sodium chloride at a fraction size of 2 mL. The fraction was eluted and fractionated, and the fraction was analyzed by SDS-PAGE in the same manner as in Example 3.

  In FIG. 20, lane 1 is a molecular weight marker protein, lane 2 is a soluble fraction before fractionation with a nickel chelate resin, lane 3 is a pass-through fraction, lanes 4 to 8 contain 0.2 M imidazole and 0.3 M sodium chloride. Fractions eluted with 0.05 M Tris-HCl buffer (pH 8.0), and FcBPf51H (molecular weight 131 kDa) was detected at the position indicated by the arrow in lane 6.

Example 21 FcBPf51H hydrolyzed with enterokinase
The fraction containing FcBPf51H obtained in Example 20 was dialyzed in 0.05 M Tris-HCl buffer (pH 8.0) containing 0.2 M sodium chloride. The FcBPf51H solution after dialysis was determined by colorimetric determination with a protein assay (trade name, Bio-Rad) using γ-globulin as a standard protein. After adding 1/500 volume of 1M calcium chloride to the FcBPf51H solution (0.24 mg / mL), add 1 / 10,000 wt. Of enterokinase of the protein mass (weight) contained in each solution and Incubated with

  A part was sampled after 18 hours, 64 hours and 112 hours after incubation, and 1/20 volume of 0.02 M N-α-Tosyl-L-lysine chloromethyl ketone hydrochloride was mixed as an inhibitor of the reaction.

  A portion of each solution was analyzed by Western blotting and enzyme immunoassay.

  For Western blotting, an HRP-labeled rabbit anti-6-His (Bethyl) was used.

  In the enzyme immunoassay, 10 μg / mL human immunoglobulin (gamma globulin) (product name, manufactured by Chemical and Serum Therapy Research Institute), 0.05 M Tris-HCl buffer (pH 8.0) per well was added to a 96-well immunoplate. Add 100 μL, incubate at 4 ° C. for 18 hours, rinse with TBS-T to separate B / F, add 200 μL of BSA / TBS-T per well, and incubate at 30 ° C. for 2 hours. After blocking.

The blocked plate was rinsed with TBS-T and subjected to B / F separation. A sample prepared by diluting with BSA / TBS-T was added at 100 μL per well and incubated at 30 ° C. for 1 hour. Then, 100 μL of HRP-labeled anti-6xHis antibody (Bethyl) diluted 5000 times with BSA / TBS-T was added per well and incubated at 30 ° C. for 1 hour. After the B / F separation, 100 μL of TMB Microwell Peroxidase Substrate (2-Component System) (product name, Kirkegaard & Perry Laboratories) was added to each well and reacted at room temperature for 30 minutes, and 1M phosphoric acid aqueous solution was reacted at room temperature for 30 minutes. After stopping the reaction by adding 50 μL per well, the absorbance (OD ₄₅₀ ) at a wavelength of 450 nm was measured.

  In FIG. 21, lane 1 of Western blotting is a molecular weight marker protein, samples of lanes 2 to 5 are not added with enterokinase, lanes 6 to 8 are samples to which enterokinase is added, and lane 2 is before incubation. Lanes 3 and 6 are the results of analyzing the sample after 18 hours of incubation, lanes 4 and 7 are the samples after 64 hours of incubation, and lanes 5 and 8 are the results of analyzing the samples after 112 hours of incubation.

  As shown in FIG. 21, FcBPf51H almost disappeared in the 112-hour sample (lane 8) after addition of enterokinase. Further, in the sample to which enterokinase was added, a product (protein) band that was considered to have been hydrolyzed with the amino acid sequence serving as a substrate to remove the T7 RNA polymerase portion was detected.

  On the other hand, as shown in FIG. 22, from the results of FcBPf51H (64h-) incubated for 64 hours without the addition of enterokinase and FcBPf51H (112h-) incubated for 112 hours without the addition of enterokinase, It was found to adsorb to human immunoglobulin depending on the concentration.

  In addition, FcBPf51H (64h +) incubated for 64 hours with the addition of enterokinase in FIG. 22 and FcBPf51H (112h +) incubated for 112 hours with the addition of enterokinase were hydrolyzed with enterokinase to remove the T7 RNA polymerase portion. It was found that the protein also adsorbed to human immunoglobulin depending on the concentration.

Example 22 Construction of Plasmid pCDF20-hGHbpf51H Human Growth Hormone Receptor (SEQ ID NO: 5) via 7 residues of Gly-Ser-Asp-Asp-Asp-Asp-Lys at the carboxyl terminus of T7 RNA polymerase (SEQ ID NO: 1) A polynucleotide (SEQ ID NO: 69) encoding a fusion protein (hGHbpf51H, SEQ ID NO: 68, FIG. 23) in which a polypeptide consisting of positions 1 to 238 is linked, followed by 6 histidines. Plasmid pCDF20-hGHbpf51H (FIG. 24) was prepared.

  First, PCR of the first stage was performed using the plasmid pET-hGHbp258 described in Japanese Patent Application No. 2009-025901 as a template, using the primer set of SEQ ID NO: 70 and SEQ ID NO: 71, and a 0.7 kbp DNA ( DNA-22)) was obtained.

  Next, the second stage PCR is performed using DNA-22 as a template and the primer set of SEQ ID NO: 71 and SEQ ID NO: 72 to obtain 0.8 kbp of DNA, which is digested with restriction enzymes BglII and BlnI. Digestion yielded 0.8 kbp DNA (referred to as DNA-23). The DNA-23 was ligated with 5.9 kbp DNA obtained by digesting the plasmid pCDF20-EPObpf60H with the restriction enzymes BamHI and BlnI, and cyclized to prepare a plasmid.

  E. coli JM109 was transformed with the plasmid and cultured in LB / Carb agar medium to obtain E. coli JM109 / pCDF20-hGHbpf51H having the target plasmid.

Example 23 Adsorption of fusion protein hGHbpf51H to human growth hormone E. coli JM109 / pCDF20-hGHbpf51H was inoculated into 2xYT / Carb medium and cultured at 28 ° C for 16 hours, and the bacterial density (OD ₆₀₀ ) was 4. 10 mL of the culture solution of 2 was obtained.

E. coli JM109 / pCDF20-hGHbpf51H was collected from the culture by centrifugation. The obtained bacteria were suspended in 0.05 M Tris-HCl buffer (pH 8.0) containing 1 mL of 0.001 M EDTA, and then centrifuged to wash the bacteria. Next, the bacteria were washed with 1 mL of 0.05 M Tris-HCl buffer (pH 8.0). The bacteria were suspended in 0.1 mg / mL egg white lysozyme, 0.05 M Tris-HCl buffer (pH 8.0) so that the value of the bacterial density (OD ₆₀₀ ) was 36. . The suspension was incubated at 0 ° C. for 30 minutes and then frozen at −80 ° C. After thawing it, it was subjected to an ultrasonic crusher, and the liquid obtained by crushing was centrifuged to obtain a soluble fraction.

  It was examined by enzyme immunoassay that hGHbpf51H contained in the soluble fraction was adsorbed to human growth hormone.

  Enzyme immunoassay was performed by adding 1 μg / mL human growth hormone to 96-well immunoplate, 100 μL of 0.05 M Tris-HCl buffer (pH 8.0) per well, and incubating at 4 ° C. for 18 hours, followed by TBS-T. B / F separation was performed by rinsing, and then 200 μL of BSA / TBS-T was added per well, followed by incubation at 30 ° C. for 2 hours for blocking.

  The blocked plate was rinsed with TBS-T and subjected to B / F separation. A sample prepared by diluting with BSA / TBS-T was added at 100 μL per well and incubated at 30 ° C. for 1 hour. separated. Subsequently, 100 μL of 0.5 μg / mL mouse anti-human growth hormone receptor antibody (IgG2b) was added per well, incubated at 30 ° C. for 1 hour, B / F separated, and further 0.25 μg / mL HRP-labeled rabbit anti-mouse 100 μL of IgG2b antibody was added per well and incubated at 30 ° C. for 1 hour. After B / F separation, 100 μL of TMB Microwell Peroxidase Substrate (2-Component System) (product name, Kirkegaard & Perry Laboratories) was added to each well for 90 seconds, and 1M phosphoric acid solution was reacted at room temperature for 90 seconds. After stopping the reaction by adding 50 μL per well, absorbance (OD450) at a wavelength of 450 nm was measured.

  As shown in FIG. 25, it was found that it adsorbs to human growth hormone depending on the concentration of hGHbpf51H.

Claims (17)

  A T7 RNA polymerase fusion protein in which a heterologous protein is fused to the carboxyl terminus of T7 RNA polymerase represented by SEQ ID NO: 1 directly or via a linker consisting of one or more amino acids.   The heterologous protein is a polypeptide derived from a vertebrate or a polypeptide obtained by substituting and / or deleting and / or adding one or more amino acids of a polypeptide derived from a vertebrate to another amino acid. The T7 RNA polymerase fusion protein according to claim 1, wherein the protein is a fusion protein.   The T7 RNA polymerase fusion protein according to claim 1 or 2, wherein the heterologous protein comprises a peptide comprising an additional sequence for protein purification.   The T7 RNA polymerase fusion protein according to claim 3, wherein the peptide consisting of the additional sequence for protein purification is selected from the group consisting of polyhistidine, S-peptide, and c-myc peptide.   Vertebrate-derived polypeptide is a polypeptide consisting of positions 1 to 225 of the human erythropoietin receptor represented by SEQ ID NO: 2, human erythropoietin represented by SEQ ID NO: 3, and positions 1 to 274 of FcγRI represented by SEQ ID NO: 4. The T7 RNA polymerase fusion protein according to any one of claims 1 to 4, wherein the T7 RNA polymerase fusion protein is a polypeptide consisting of 1 or 238 of the human growth hormone receptor represented by SEQ ID NO: 5.   The T7 RNA polymerase fusion protein according to any one of claims 1 to 5, wherein the linker is a linker having an amino acid sequence serving as a substrate for one or more types of proteases.   The T7 RNA polymerase fusion protein according to claim 6, wherein the protease is enterokinase, thrombin, activated blood coagulation factor X, urokinase or HRV 3C protease.   6. The linker according to any one of claims 1 to 5, wherein the linker is a linker having an amino acid sequence serving as a substrate for any of enterokinase, thrombin, activated blood coagulation factor X, urokinase or HRV 3C protease. T7 RNA polymerase fusion protein.   The amino acid sequence serving as the protease substrate is Asp-Asp-Asp-Asp-Lys, Lue-Val-Pro-Arg, Ile-Glu-Gly-Arg, Pro-Gly-Arg or Leu-Glu-Val-Leu-Phe The T7 RNA polymerase fusion protein according to claim 6, which is -Gln-Gly-Pro.   The linker is Asp-Asp-Asp-Asp-Lys, Lue-Val-Pro-Arg, Ile-Glu-Gly-Arg, Pro-Gly-Arg or Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro The T7 RNA polymerase fusion protein according to any one of claims 1 to 5, which is a linker having any one of the amino acid sequences.   A polynucleotide encoding the T7 RNA polymerase fusion protein according to any one of claims 1 to 10.   A bacterium of the genus Escherichia that produces the T7 RNA polymerase fusion protein according to any one of claims 1 to 10.   A method for producing a heterologous protein, comprising hydrolyzing the T7 RNA polymerase fusion protein according to any one of claims 1 to 10 with a protease.   The method according to claim 13, wherein the protease is enterokinase, thrombin, activated blood coagulation factor X, urokinase or HRV 3C protease.   The method for producing a heterologous protein according to claim 13 or 14, wherein the T7 RNA polymerase fusion protein is produced using a bacterium of the genus Escherichia according to claim 12.   The production method according to claim 15, wherein Cibacron Blue fixing resin is used.   A T7 RNA polymerase fusion protein according to any one of claims 1 to 10 or a heterologous protein produced by hydrolyzing the T7 RNA polymerase fusion protein according to any one of claims 1 to 8 with a protease, The method for producing a heterologous protein according to any one of claims 13 to 16, wherein the protein is separated using a protein.