JP5637180B2 - Method for producing protein as soluble protein - Google Patents

Description

The present invention relates to a method for producing a target protein as a soluble protein. More specifically, the present invention relates to a method for producing a target protein as a soluble protein using a secretory signal peptide and a peptide rich in basic amino acids.

So far, many recombinant protein expression systems such as recombinant protein expression systems and cell-free translation systems in hosts such as bacteria, yeast, insects, transgenic animals and transgenic plants have been developed. Among them, Escherichia coli can be easily grown at a high density and has been widely used as a heterologous protein expression system because of the progress of research on host vector systems.
On the other hand, when the target protein is expressed in these recombinant protein expression systems, the expressed protein cannot be folded correctly, so that the original function of the protein cannot be expressed, or insoluble aggregates called inclusion bodies are formed. Often formed. In such a case, for example, even if the inclusion body is denatured (solubilized) and then refolded, a protein that is normally folded and has the original function is not necessarily obtained. Moreover, even if a protein that expresses the original function is obtained, a satisfactory recovery rate is often not obtained.
In such a background, a method for suppressing the formation of inclusion bodies of the expressed recombinant target protein has not been established so far. As an alternative, an attempt has been made to express a soluble protein by fusing a highly soluble maltose-binding protein having a molecular weight of 40,000 or glutathione S-transferase (GST) and an insoluble target protein (Non-patent Document 1: Fox, JDand Waugh, DS (2003) Maltose-binding protein as a solubility e
nhancer. Methods Mol. Biol. 205: 99-117, Non-Patent Document 2: Ausubel, FM et al. eds.
1996) Current Protocols in Molecular Biology, Vol. 2, 16.0.1). However, there are problems that the soluble protein does not exhibit the original activity, and that when the maltose binding protein or GST is deleted, the target protein is insolubilized.

Renilla luciferin binding protein (hereinafter abbreviated as RLBP) is Renilla (Re
calcium-induced luciferin-binding protein (Calcium-trigger) from nilla reniformis
ed luciferin-bindihg protein). RLBP is a noncovalent complex of apoprotein (apoRLBP) and coelenterazine (luciferin) (Non-patent Document 3).
: J. Biol. Chem. 254, 769-780 (1979)). When RLBP binds to calcium ions,
Coelenterazine is released. The released coelenterazine is used for the luciferin-luciferase luminescence reaction by Renilla luciferase, Evil luciferase, Gaussia luciferase and the like using coelenterazine as the luminescent substrate.

By fusing the E. coli L-asparaginase gene with a pelB leader sequence and an N-terminal 6 × histidine tag, recombinant E. coli L-asparaginase is expressed in culture filtrate outside the cell and purified by Ni-NTA affinity chromatography. A method has been reported (Non-patent Document 4: Protein Express. Purif. 38, 29-36 (2004)).
Fox, JDand Waugh, DS (2003) Maltose-binding protein as a solubility enhancer. Methods Mol. Biol. 205: 99-117 Ausubel, FM et al. Eds. (1996) Current Protocols in Molecular Biology, Vol. 2, 16.0.1 J. Biol. Chem. 254, 769-780 (1979) Protein Express. Purif. 38, 29-36 (2004)

In the above situation, it is strongly desired in the industry to produce a target protein as a soluble protein using a recombinant protein expression system for the production of useful proteins. There is also a strong demand in research fields such as protein function and structure research.

As a result of intensive research to solve the above problems, the present inventors combined a polynucleotide encoding a signal peptide and a polynucleotide encoding a polypeptide rich in basic amino acids with a polynucleotide encoding a target protein. It has been found that when a target protein is expressed in a gram-negative bacterium using an expression vector containing the prepared polynucleotide, the target protein is expressed as a soluble protein. Furthermore, it was confirmed that the target protein thus obtained has an original function. As a result of further studies based on these findings, the present invention has been completed.

That is, the present invention
(1) A protein is expressed using a polynucleotide containing a polynucleotide encoding a secretory signal peptide, a polynucleotide encoding a polypeptide rich in basic amino acids, and a polynucleotide encoding a target protein in this order. A method for producing the target protein as a soluble protein,
(2) The method according to (1) above, wherein the protein is expressed in a host cell,
(3) The method according to (2) above, wherein the host cell is a gram-negative bacterium,
(4) The method according to (3) above, wherein the gram-negative bacterium is an Escherichia coli bacterium,
(5) The above (wherein the secretory signal peptide is a secretory signal peptide derived from Gram-negative bacteria (
The method according to any one of 1) to (4),
(6) The method according to (5) above, wherein the secretion signal peptide derived from Gram-negative bacteria is a secretion signal peptide derived from facultative anaerobic bacilli.
(7) The method according to (5) above, wherein the signal peptide derived from Gram-negative bacteria is a secretion signal peptide derived from outer membrane protein A of E. coli (OmpA) or a secretion signal peptide derived from cholera toxin cholera toxin.
(8) The method according to any one of (1) to (7) above, wherein the polypeptide rich in basic amino acids is a polypeptide comprising 5 to 12 amino acid residues,
(9) The method according to any one of (1) to (8) above, wherein the basic amino acid content in the polypeptide rich in basic amino acids is 60% or more,
(10) The method according to any one of (1) to (9) above, wherein the basic amino acid in the polypeptide rich in basic amino acids is selected from histidine, arginine and lysine,
(11) The above-mentioned (1) to (9), wherein the polypeptide rich in basic amino acids is polyhistidine.
)
(12) The method according to any one of (1) to (11) above, wherein the expression of the target protein is performed using an expression vector containing a polynucleotide encoding the target protein.
(13) A polynucleotide comprising a polynucleotide encoding a secretion signal peptide of a Gram-negative bacterium, a polynucleotide encoding a polypeptide comprising 5 to 12 basic amino acid residues, and a polynucleotide encoding a target protein A method for producing the target protein as a soluble protein, characterized by expressing the protein in a gram-negative bacterium using nucleotides,
(14) characterized in that a protein is expressed in Escherichia coli using a polynucleotide containing a polynucleotide encoding OmpA, a polynucleotide encoding polyhistidine, and a polynucleotide encoding a target protein. A method for producing the target protein as a soluble protein,
(15) The method according to any one of (1) to (14) above, wherein the target protein is a heterologous protein,
(16) The above (1), wherein the target protein is any one selected from the group consisting of apoRLBP, apoaequorin, apocritin, apoovelin, and apomytrocomin.
To (14)
(17) The method according to (16) above, wherein the target protein is apoRLBP,
(18) A polynucleotide comprising a polynucleotide encoding a secretion signal peptide of a Gram-negative bacterium, a polynucleotide encoding a polypeptide consisting of 5 to 12 basic amino acid residues, and a polynucleotide encoding apoRLBP A method for producing apoRLBP, characterized in that a protein is expressed in a Gram-negative bacterium using nucleotides;
(19) A protein is expressed in Escherichia coli using a polynucleotide containing a polynucleotide encoding OmpA, a polynucleotide encoding polyhistidine, and a polynucleotide encoding apoRLBP, and the expressed protein is periplasm Apo RLBP manufacturing method, characterized by being accumulated in space,
(20) A method for producing RLBP, comprising contacting apoRLBP produced by the production method according to any of (17) to (19) above with coelenterazine or a derivative thereof,
(21) A coelenterazine or a derivative thereof, characterized in that RLBP is prepared by contacting apoRLBP produced by the production method according to any of (17) to (19) above with coelenterazine or a derivative thereof. Preservation method,
(22) RLBP comprising apoRLBP and coelenterazine or a derivative thereof produced by the production method according to any of (17) to (19) above,
(23) (a) a first coding region encoding a secretory signal peptide,
(B) a second coding region encoding a polypeptide rich in basic amino acids, and (c) a third coding region encoding a protein of interest can be inserted at least one
An expression vector comprising one restriction enzyme site,
(23a) The method according to (23) above, wherein the first coding region is effectively linked to a promoter,
(23b) The method according to (23) above, wherein the first coding region, the second coding region, and the restriction enzyme site are in the same reading frame.
(23c) The method according to (23) above, wherein the second coding region is downstream of the first coding region, and the restriction enzyme site is downstream of the second coding region,
(23d) The method according to (23) above, wherein the restriction enzyme site is a multicloning site,
(24) The expression vector according to the above (23), wherein the secretion signal peptide is a secretion signal peptide derived from Gram-negative bacteria,
(25) The expression vector according to the above (24), wherein the secretion signal peptide derived from Gram-negative bacteria is a secretion signal peptide derived from facultative anaerobic bacilli.
(26) The expression vector according to (24), wherein the secretory signal peptide derived from Gram-negative bacteria is a secretory signal peptide derived from outer membrane protein A of E. coli (OmpA) or a secretory signal peptide derived from cholera toxin cholera toxin,
(27) The expression vector according to any one of (23) to (26), wherein the polypeptide rich in basic amino acids is a polypeptide consisting of 5 to 12 amino acid residues,
(28) The basic amino acid content in the polypeptide rich in basic amino acids is 60%.
The expression vector according to any one of (23) to (27) above,
(29) The expression vector according to any one of (23) to (28), wherein the basic amino acid in the polypeptide rich in basic amino acids is selected from histidine, arginine and lysine,
(30) The above-mentioned (23) to (23), wherein the polypeptide rich in basic amino acids is polyhistidine.
27) the expression vector according to any one of
(31) (a) a first coding region encoding a secretory signal peptide derived from a Gram-negative bacterium,
(B) a second coding region encoding a polypeptide consisting of 5 to 12 basic amino acid residues, and (c) a third coding region encoding a target protein can be inserted at least 1
An expression vector comprising one restriction enzyme site,
(32) (a) a first code region encoding OmpA,
(B) a second coding region encoding polyhistidine and (c) a third coding region encoding a protein of interest can be inserted at least one
An expression vector or the like containing one restriction enzyme site is provided.

According to the present invention, when producing a target protein using a recombinant protein expression system,
Since the target protein can be produced as a soluble protein, it is not necessary to denature (solubilize) the target protein. Therefore, according to the present invention, the target protein can be obtained efficiently and at a high recovery rate.
Therefore, the present invention is extremely useful as a method for producing useful proteins and proteins such as proteins to be analyzed for functions and structures.

The present invention expresses a protein using a polynucleotide containing a polynucleotide encoding a secretory signal peptide, a polynucleotide encoding a polypeptide rich in basic amino acids, and a polynucleotide encoding a target protein. Provided are a method for producing the target protein, an expression vector that can be used for the production method, and the like. According to the production method of the present invention, by expressing a target protein in a state fused with a secretory signal peptide and a polypeptide rich in basic amino acids, the target protein is not normally folded and forms an inclusion body or the like. It is possible to prevent the target protein from being solubilized and to be produced as a protein having an original function that is normally folded.
Hereinafter, the production method of the target protein of the present invention will be described in detail.

1. Production method of target protein The present invention comprises (1) a polynucleotide encoding a secretory signal peptide, (2) a polynucleotide encoding a polypeptide rich in basic amino acids, and (3) a polynucleotide encoding a target protein. Are produced using a polynucleotide containing these in this order, and a method for producing a target protein is provided. This will be described in more detail below.
(1) Secretory signal peptide The secretory signal peptide means a peptide region that plays a role of allowing a protein or polypeptide bound to the secretory signal peptide to permeate through the cell membrane. The amino acid sequences of such secretory signal peptides and the nucleic acid sequences that encode them are well known and reported in the art (eg von Heijine G (1988) Biochim. Biohys. Acra 9
47: 307-333, von Heijine G (1990) J. Membr. Biol. 115: 195-201.
The secretory signal peptide usually has an amino acid sequence consisting of about 10 to 50 amino acid residues, and many of the amino acid residues (usually about 55 to 60%) are hydrophobic. The secretory signal peptide used in the present invention is preferably derived from a prokaryotic organism, more preferably from a gram-negative bacterium, more preferably from a facultative anaerobic gonococcus, particularly preferably from the genus Escherichia, Pseudomonas aeruginosa, Salmonella or cholera. Secretion signal peptides derived from bacteria of the genus Mycobacterium are preferred. Examples of secretory signal peptides include von Heijine G & Abrahmsen, L. (1989) FEB
S Lett. 244: 439-446. Tjalsma, H. et al. (2000) Microbiol. Mol Rev. 515-547 and the like are mentioned, and in particular, a secretion signal peptide derived from outer membrane protein A of Escherichia coli. (OmpA) (Ghrayeb, J. et al. (1984) EMBO J. 3: 2437-2442), a cholera toxin-derived cholera toxin-derived secretory signal peptide, and the like are preferable.
The secretory signal peptide used in the present invention may be a mutant as long as it has an activity of allowing the protein or polypeptide bound to the secretory signal peptide to permeate the cell membrane. A variant includes a protein or polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of a secretory signal peptide and bound to the secretory signal peptide. And peptides having an activity of permeabilizing the cell membrane. Examples of such peptides include 1 to 10, 1 to 9, 1 to 8, 1 and 1 in the amino acid sequence of the secretion signal peptide.
~ 7, 1-6 (1 ~ several), 1-5, 1-4, 1-3, 1-2, 1 amino acid residue deleted, substituted, inserted and / or Or the peptide which consists of an added amino acid sequence and has the activity which permeate | transmits a protein or polypeptide couple | bonded with the said peptide to a cell membrane is mentioned. In general, the smaller the number of amino acid residue deletions, substitutions, insertions and / or additions, the better. Two or more of deletion, substitution, insertion and addition may occur simultaneously. Such proteins also include the amino acid sequence of the target protein and more than 80%, 85
% Or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more 99
A peptide having an amino acid sequence having an identity of .8% or more and 99.9% or more and having an activity of permeating the protein or polypeptide bound to the peptide through the cell membrane can be mentioned. In general, the larger the homology value, the better.
Secretory signal peptides generally have a cleavage site that is cleaved by a signal peptidase upon membrane permeation. The secretory signal peptide used in the present invention may not have a cleavage site by a signal peptidase, but preferably has a cleavage site. As the cleavage site, one that can be cleaved by a secretory signal peptidase of a host (for example, Escherichia coli) used for expression of the target protein is preferably used.

(2) Polypeptide rich in basic amino acid The polypeptide rich in basic amino acid used in the present invention contains a basic amino acid and dissolves the target protein when fused and expressed on the N-terminal side of the target protein. It may be anything that can increase the property. Examples of the polypeptide rich in basic amino acids include, for example, a polypeptide having an isoelectric point on the alkaline side of physiological pH, and a ratio (content ratio) of the number of basic amino acid residues to all amino acid residues constituting the polypeptide. And the like, which are 30% or more. The content of basic amino acid residues is preferably 60% or more,
Further, it is preferably 80% or more, and particularly preferably 100%.
The number of amino acid residues contained in the polypeptide rich in basic amino acids used in the present invention is preferably 3 to 20 residues, more preferably 5 to 12 residues, particularly 5 to 8 residues. It is preferably a group, especially 6 residues.
A basic amino acid means an amino acid having a basic side chain. Examples of basic amino acids include histidine, arginine, lysine and the like. As the basic amino acid used in the present invention, histidine, arginine and lysine are preferable, and histidine is particularly preferable.
The ratio (content ratio) of the number of histidine residues to the basic amino acid residues contained in the polypeptide rich in basic amino acids is preferably 60% or more, more preferably 80% or more, particularly It is preferably 100%.
As the polypeptide rich in basic amino acids used in the present invention, polyhistidine is particularly preferable. The number of histidine residues contained in polyhistidine is preferably 5 to 12 residues, more preferably 5 to 8 residues, and particularly preferably 6 residues (histidine hexamer).

(3) Target protein There is no restriction | limiting in particular in the target protein in the manufacturing method of this invention. For example, a protein that easily forms an inclusion body when expressed in a recombinant protein expression system can be produced as a soluble protein, and thus can be preferably used. The target protein may be an apoprotein, that is, a protein portion of a holoprotein. Examples of the apoprotein include apoRLBP (see FEBS Lett. 268, 287-290 (1990), etc.), apoaequorin (see Proc. Natl. Acad. Sci. USA 82, 3154-3158 (1985), etc.), apocritin (See FEBS Lett. 315, 343-346 (1993), etc.), Apomaitrocomin (FEBS Lett. 333,
301-305 (1993), etc.) and apooverin (see Gene 153,273-274 (1995), etc.). The amino acid sequence of apoRLBP is shown in SEQ ID NO: 1.
In addition, proteins (viral antigens) such as coat protein, core protein, protease, reverse transcriptase, integrase encoded by pathogenic virus genomes such as hepatitis B virus, hepatitis C virus, HIV, influenza; antibody Fab, (Fab) ₂
Platelet derived growth factor (PDGF), stem cell growth factor (SCF), hepatocyte growth factor (HG)
F), growth factors such as transforming growth factor (TGF), nerve growth factor (NGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF); tumor necrosis factor , Interferon, interleukin and other cytokines; erythropoietin, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, thrombopoietin and other hematopoietic factors; luteinizing hormone releasing hormone (LH-RH), thyroid stimulating hormone Release hormone (TRH), insulin, somatostatin, growth hormone, prolactin, corticotropin (ACTH)
), Melanocyte stimulating hormone (MSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), vasopressin, oxytocin, calcitonin, parathyroid hormone (PTH), glucagon, gastrin, secretin, pan Peptide hormones such as cleosaimine, cholecystokinin, angiotensin, human placental lactogen, human chorionic gonadotropin (HCG), cerulein, motilin; analgesic peptides such as enkephalin, endorphin, dinorphine, kyotorphin; superoxide dismutase (SOD), urokinase, tissue plasminogen activator (TPA), asparaginase, kallikrein and other enzymes; bombesin,
Peptide neurotransmitters such as neurotensin, bradykinin, substance P; albumin; collagen; proinsulin; renin; α1 antitrypsin and the like may be mentioned, but not limited thereto.

The target protein of the present invention includes mutants of the above proteins. Examples of such a mutant include a protein having an amino acid sequence in which one or a plurality of amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of the protein and having the same activity as the target protein. Is also included. Examples of such proteins include 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, and 1 to 40 in the amino acid sequence of the protein. 1-30, 1-20, 1-10, 1-9, 1-8,
1-7, 1-6 (1 to several), 1-5, 1-4, 1-3, 1-2, 1 amino acid residue deleted, substituted, inserted and And / or a protein having an added amino acid sequence and having the same activity as the target protein. Deletion, substitution of the above amino acid residues,
In general, the smaller the number of insertions and / or additions, the better. Two or more of deletion, substitution, insertion and addition may occur simultaneously. In this specification, apoRLBP means not only a protein having the amino acid sequence represented by SEQ ID NO: 1, but also a variant thereof.

The target protein of the present invention includes “partial peptides” of the target protein. Examples of the partial peptide of the protein include partial peptides consisting of a part of the amino acid sequence of the target protein, and preferably have the same activity as that of the target protein. Examples thereof include a polypeptide having an amino acid sequence composed of at least 20, preferably at least 50 amino acid residues in the amino acid sequence of the target protein. Preferably, these polypeptides contain an amino acid sequence corresponding to the portion involved in the activity of the target protein. Moreover, the partial peptide used in the present invention is one or more of the above-described polypeptides in the amino acid sequence (for example, about 1 to 20, more preferably about 1 to 10, even more preferably 1). (About ˜5) amino acid residues may be changed by deletion, addition, substitution, or insertion.
The partial peptide used in the present invention can also be used as an antigen for antibody production.

(4) Polynucleotide used in the present invention The polynucleotide used in the present invention is any polynucleotide as long as it contains a secretory signal peptide, a polypeptide rich in basic amino acids, or a base sequence encoding a target protein. However, it is preferably DNA. As DNA,
Examples include genomic DNA, genomic DNA library, cell / tissue-derived cDNA, cell / tissue-derived cDNA library, and synthetic DNA. As an example of the polynucleotide encoding the target protein of the present invention, SEQ ID NO: 2 shows the base sequence of a synthetic DNA encoding apoRLBP.
The vector used for the library is not particularly limited and may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, from the cells and tissues described above
Reverse Transcription Po directly using the total RNA or mRNA fraction prepared
It can also be amplified by lymerase chain reaction (hereinafter abbreviated as RT-PCR method).
A polynucleotide (DNA) containing a polynucleotide encoding a secretory signal peptide used in the present invention, a polynucleotide encoding a polypeptide rich in basic amino acids, and a polynucleotide encoding a target protein is a target protein. There may be further provided a polynucleotide encoding a cleavable peptide linker between the encoding polynucleotide and a polynucleotide encoding a secretory signal peptide or a polynucleotide encoding a polypeptide rich in basic amino acids. . A cleavable peptide linker means a peptide sequence that can be cleaved by an enzymatic or chemical cleaving substance. Many peptide sequences that are cleaved by enzymes (proteases) or chemicals are known (eg, Harlow and Lane, Antibodies: A Laboratory Manual, Co
ld Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988); Walsh, Protein
s Biochemistry and Biotechnology, John Wiley & Sons, LTD., West Sussex, England
(See 2002). A cleaving substance is a chemical substance or enzyme that recognizes a cleaving site in a polypeptide and splits the polypeptide into two polypeptides by cleaving a bond in the polypeptide. Examples of the cleaving substance include chemical substances and proteases.

(5) Production of target protein Specifically, the target protein of the present invention includes, for example, a polynucleotide encoding a secretory signal peptide, a polynucleotide encoding a polypeptide rich in basic amino acids, and a polynucleotide encoding the target protein. An expression vector obtained by linking (introducing) a polynucleotide (DNA) containing nucleotides in this order (hereinafter, sometimes referred to as “polynucleotide used in the present invention” in the description of the production of the target protein). It can be produced by expressing a target protein and separating and purifying the generated target protein. Expression of the target protein using the expression vector can be performed in a host cell, a protein expression system such as a cell-free translation system. The target protein is preferably expressed in a host cell (transformant) transformed by introducing the expression vector.
The transformant is cultured under conditions allowing expression of the polynucleotide (DNA) used in the present invention linked (introduced) to the introduced expression vector, and the target protein is purified / accumulated and separated / purified. Can be manufactured. As the host cell, Gram-negative bacteria are preferable, and Escherichia coli is particularly preferably used. When a Gram-negative bacterium is used as a host cell, the expressed target protein preferably permeates the inner membrane by a secretory signal peptide, passes through the periplasmic space or outer membrane, and is secreted into the culture supernatant. . Here, the target protein of the present invention may be a heterologous protein. A heterologous protein means a protein that is not naturally produced in a protein expression system such as a host cell.
Hereinafter, the method for producing the target protein of the present invention will be described in more detail.

(Creation of expression vector)
The expression vector used in the present invention contains the polynucleotide used in the present invention. The recombinant vector of the present invention can be obtained by ligating (inserting) the polynucleotide (DNA) used in the present invention into an appropriate vector. More specifically, the purified polynucleotide (DNA) used in the present invention is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multiple cloning site of an appropriate vector, and ligated to the vector. Can be obtained. The vector for inserting the polynucleotide used in the present invention is not particularly limited as long as it can replicate in the host, and examples thereof include plasmids, bacteriophages and the like. Examples of plasmids include plasmids derived from E. coli (eg, pUC8, pUC118, pUC119, pBR322, pBR325, etc.). Examples of the bacteriophage include λ phage.

The polynucleotide of the present invention is usually operably linked downstream of a promoter in an appropriate vector. The promoter used is preferably one that can be expressed in the host. More specifically, for example, when the host to be transformed is an Escherichia coli bacterium, lpp promoter, Trp promoter, T7 promoter, lac promoter, recA promoter, λPL promoter and the like are preferable.
In addition to the above, the recombinant vector of the present invention may optionally include a ribosome binding sequence (SD sequence).
Those containing a selection marker or the like can be used. As a selection marker,
Examples thereof include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, and the like.

(Creation of transformants)
The transformant used in the present invention includes a polynucleotide (DNA) used in the present invention.
The expression vector used in the present invention has been introduced so that can be expressed. A transformant can be prepared by introducing an expression vector containing the polynucleotide (DNA) used in the present invention obtained as described above into a suitable host. The host is not particularly limited as long as it can express the polynucleotide (DNA) used in the present invention, and examples thereof include Gram-negative bacteria. As gram negative bacteria,
For example, facultative anaerobic bacilli and the like can be mentioned. Examples of facultative anaerobic bacilli include bacteria of the genus Escherichia coli, Pseudomonas aeruginosa, or Salmonella. Examples of bacteria belonging to the genus Escherichia coli include Escherichia coli. Examples of bacteria belonging to the genus Pseudomonas aeruginosa include Pseudomonas aeruginosa. Examples of bacteria belonging to the genus Salmonella include Salmonella. As the host, Gram-negative bacteria are preferable, facultative anaerobic bacilli are more preferable, bacteria of the genus Escherichia are preferable, and Escherichia coli is particularly preferable.

The method for introducing the expression vector into the host and the transformation method using the vector can be performed by various general methods. Examples of methods for introducing an expression vector into a host cell include the calcium phosphate method (Virology, 52, 456-457 (1973)) and the electroporation method (EMBO).
J., 1, 841-845 (1982)). As a method for transforming E. coli bacteria,
Examples thereof include the methods described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982) and the like. Examples of a method for transforming Pseudomonas aeruginosa bacteria include electroporation. As a method for transforming Salmonella bacteria,
For example, there is an electroporation method.
Thus, a transformant transformed with an expression vector containing the polynucleotide (DNA) used in the present invention can be obtained.
(Culture of transformants)
The transformant used in the present invention can be cultured according to a usual method used for host culture. By the culture, the target protein is produced by the transformant, and the target protein is accumulated in the periplasmic space or the culture solution.

As a medium for culturing a transformant whose host is a bacterium of the genus Escherichia coli, Pseudomonas aeruginosa or Salmonella, it contains a carbon source, a nitrogen source, inorganic salts, etc. necessary for the growth of the transformant. Any natural or synthetic medium may be used as long as the medium can be efficiently cultured. As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate,
In addition to inorganic or organic acid ammonium salts such as ammonium acetate and ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, and the like are used. Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary. When cultivating a transformant transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a transformant transformed with an expression vector using the Lac promoter, transformation by transforming isopropyl-β-D-thiogalactopyranoside (IPTG) with an expression vector using the trp promoter When culturing the body, indoleacrylic acid (IAA) or the like may be added to the medium.

When the host is a bacterium of the genus Escherichia, Pseudomonas or Salmonella, the culture is usually about 15 to 4
Carry out at 3 ° C. for about 3 to 24 hours, and if necessary, add aeration and stirring.

(Separation and purification of target protein)
The target protein of the present invention can be obtained by separating and purifying the target protein from the culture. Here, the culture means any of a culture solution, cultured cells or cultured cells, or disrupted cultured cells or cultured cells. Separation of target protein
Purification can be performed according to a usual method.

Specifically, when the target protein is accumulated in the periplasmic space, an extract containing the target protein can be obtained by an ordinary method (for example, osmotic shock method) after the end of the culture. When the target protein accumulates in the culture solution, after completion of the culture, the cells or cells and the culture supernatant are separated by a usual method (for example, centrifugation, filtration, etc.), thereby A culture supernatant containing can be obtained. When the target protein is accumulated in cultured cells or cultured cells, after culturing, the cells or cells are disrupted by a normal method (for example, ultrasound, lysozyme, freeze-thawing, etc.), and then the normal method ( For example, the target protein extract can be obtained by centrifugation, filtration and the like.

Purification of the target protein contained in the extract or culture supernatant thus obtained can be performed according to a normal separation / purification method. Examples of separation / purification methods include:
Ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography,
Affinity chromatography, reverse phase high performance liquid chromatography, dialysis, ultrafiltration, etc. can be used alone or in appropriate combination.

When the target protein is an apoprotein, the holoprotein can be produced by an ordinary method, for example, by contacting the obtained apoprotein and a non-protein component. For example, when the apoprotein obtained as described above is an apoprotein of a luciferin-binding protein that binds coelenterazine (for example, apoRLBP, apoaequorin, apocritin, etc.), the apoprotein is coelenterazine or a derivative thereof ( For example, by contacting with h-coelenterazine, e-coelenterazine, Bis-coelenterazine, etc.), a holoprotein of the luciferin-binding protein (for example,
RLBP, apoRLBP, apoaequorin, etc.) can be obtained. More specifically,
RLBP is Charbonneau, H. and Cormier, MJ Ca2 + -induced bioluminescence in
Renilla reniformis. Purification and characterization of a calcium-triggered luc
iferin-binding protein. (1979) J. Biol. Chem. 254, 769-780. Aequorin is, for example, Shimomura, O. and Inouye, S. (19
99) The in situ regeneration and extraction of recombinant aequorin from Escheri
chia coli cells and the purification of extracted aequorin.
nd Purification. 16: 91-95 and the like.
Coelenterazine and coelenterazine derivatives are unstable in a solution, but are stable in a state of being bound in a holoprotein of a luciferin-binding protein obtained by the production method of the present invention. Therefore, the apoprotein of luciferin-binding protein such as apoRLBP obtained by the production method of the present invention can be suitably used for stably storing luciferin such as coelenterazine and its derivatives.
In this specification, the luciferin-binding protein holoprotein includes a luciferin-binding protein apoprotein and luciferin (for example, coelenterazine), and further includes a luciferin-binding protein apoprotein and a luciferin derivative (for example, h- And coelenterazine derivatives such as coelenterazine, e-coelenterazine, and Bis-coelenterazine). The RLBP includes those composed of apo RLBP and coelenterazine, and further includes those composed of apo RLBP and a coelenterazine derivative.

2. Further, the present invention provides (a) a first coding region encoding a secretory signal peptide, (b)
An expression vector comprising: a second coding region encoding a polypeptide rich in basic amino acids; and (c) at least one restriction enzyme site into which a third coding region encoding a target protein can be inserted. Also provide. The expression vector of the present invention is (
c) By ligating (inserting) a polynucleotide encoding the above-mentioned target protein into at least one restriction enzyme site into which the third coding region encoding the target protein can be inserted, the above-mentioned target protein can be produced. It can be preferably used.
(A) Examples of the signal peptide encoded by the first coding region encoding the secretory signal peptide include those described in the above-described method for producing a target protein.
(B) Examples of the polypeptide rich in basic amino acid encoded by the second coding region encoding the polypeptide rich in basic amino acid include those similar to those described in the above-mentioned method for producing a target protein. It is done.
(C) At least one restriction enzyme site capable of inserting the third coding region encoding the target protein is a polynucleotide having a restriction enzyme recognition site capable of inserting the third coding region encoding the target protein. It contains. The restriction enzyme site is not particularly limited as long as the third coding region encoding the target protein can be inserted, but is preferably a so-called multicloning site. Restriction enzyme sites such as multicloning sites are well known and reported in the art (eg, Yanisch-Perron, C., Vieira, J. and Messing, J. Gene 33 (1985)
103-119, Improved M13 phage cloning vectors and host strains: Nucleotide sequenc
es of the M13mp18 and pUC19 vectors. See Gene 33 (1985) 103-119).
Examples of the target protein include those described in the above-described method for producing a target protein.

The expression vector of the present invention is not particularly limited as long as it can replicate in the host, for example,
Examples include plasmids and bacteriophages. Examples of plasmids include plasmids derived from E. coli (eg, pUC8, pUC118, pUC119, pBR322, pBR325, etc.). Examples of the bacteriophage include λ phage. Gram-negative bacteria are preferred as hosts used in the production of the above-mentioned target protein, facultative anaerobic bacilli are more preferred, bacteria of the genus Escherichia, Pseudomonas aeruginosa or Salmonella are preferred, especially bacteria of the genus Escherichia Therefore, the expression vector of the present invention is preferably one that can replicate in these bacteria, more preferably one that can replicate in bacteria of the genus E. coli, and particularly a plasmid derived from E. coli.

In the expression vector of the present invention, usually, the first coding region is effectively linked to a promoter. As the promoter to be used, when the host is a bacterium of the genus E. coli, lpp promoter, Trp promoter, T7 promoter, lac promoter,
RecA promoter, λPL promoter and the like are preferable. As a host used in the production of the target protein described above, a gram-negative bacterium is preferable, a facultative anaerobic gonococcus is more preferable,
Furthermore, Escherichia coli and bacteria are preferable, and Escherichia coli is particularly preferable. Therefore, promoters that can be expressed are preferably those that can be expressed in these bacteria, and those that can be expressed in bacteria of the Escherichia genus are preferable. Those that can be expressed in E. coli are preferred. More specifically, the lpp promoter, Trp promoter, T7 promoter, lac promoter, recA promoter, λPL promoter and the like are preferable.

The first coding region, the second coding region, and the restriction enzyme site are arranged so as to be in the same reading frame. Preferably, the second code region is downstream of the first code region. The restriction enzyme site is preferably downstream of the second coding region.
As the expression vector of the present invention, a vector in which a third coding region containing a polynucleotide encoding a target protein is incorporated in the restriction enzyme site is also preferable.
Furthermore, a cleavable peptide linker is encoded between at least one restriction enzyme site into which the third coding region encoding the protein can be inserted and the first coding region or the second coding region. It may have a region. The cleavable peptide linker represents the same meaning as that described in the above-mentioned method for producing a target protein. The target protein encoded by the third coding region can be cleaved by cleaving the cleavage site of the cleavable peptide linker by treatment with a protease or chemical substance.

Unless otherwise explained in the best mode and examples for carrying out the invention, J. Sambrook,
EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd
edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM A
usubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Str
A method described in a standard protocol collection such as Uhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd., or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.
The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily implement the present invention from the description of the present specification. it can. The best mode for carrying out the invention and specific examples show preferred embodiments of the present invention and are shown for illustration or description, and the present invention is not limited thereto. Not what you want. It will be apparent to those skilled in the art that various modifications can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

EXAMPLES The present invention will be described below with reference to examples, but the examples are not intended to limit the present invention.
[Materials and methods]
First, materials and methods used in this example will be described.
(1) Material Commercially available coelenterazine and coelenterazine derivatives were used. Coelenterazine (manufactured by Chisso Corporation), h-coelenterazine (manufactured by Chisso Corporation), e-coelenterazine (manufactured by Wako Pure Chemical Industries, Ltd.), Bis-coelenterazine (manufactured by Chisso Corporation).
Renilla reniformis recombinant Renilla luciferase was prepared according to literature methods (see Biochem. Biophys. Res. Commun. 233, 249-353 (1997)). The following materials were used commercially: Chelate Sepharose Fast Flow, Sephadex G25 (Super Fine Grade) (Amersham Bioscience); Imidazole, Dithiothreitol (DTT), Ethylenediaminetetraacetic acid (EDTA), Sulfuric acid Nickel hexahydrate (Wako Pure Chemical Industries).
(2) Mass spectrometry Auto FLEX with matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF-MS)
(Blue Car Daltonics) was measured according to the method described in Anal. Biochem. 316, 216-222 (2003) using sinapinic acid (Aldrich-Sigma) as a matrix.
Data were acquired in positive reflector mode. Time-mass conversion was performed by external calibration using apomyoglobin (horse, m / z = 16952.6) as a standard. The calculated molecular weight was obtained using the program ExPASy PeptideMass (http://www.expasy.ch/cgi-bin/peptide-mass.pl).
(3) Protein concentration was determined by Bradford's dye binding method (Anal. Biochem. 72, 248-254
(1976)) using a commercially available kit (BioRad) and bovine serum albumin (Pierce) as a standard substance. SDS-PAGE analysis under reducing conditions, 12% separation gel (Tefco)
Was performed according to the Laemmli method (Nature 227, 680-658 (1970)).

Reference example 1
(Construction of apoaequorin expression vector piP-HE)
An apoaequorin expression vector piP-HE having a sequence encoding OmpA and no sequence encoding a polypeptide rich in basic amino acids is known as Proc. Natl. Acad. Sci. USA.
82, 3154-3158 (1985), J. Biochem. 105, 473-477 (1989), JP-A 63-102695.
It was constructed according to the method described in the gazettes. Specifically, it was constructed as follows.
(1) Preparation of piQ8-HE The EcoRI-HindIII part of the high-copy cloning vector pUC8 was digested with the respective restriction enzymes, and cDNA obtained according to the method described in JP-A-61-135586
PiQ8-HE was prepared by subcloning the EcoRI-HindIII fragment of the aequorin cDNA obtained from clone pAQ440.
(2) Preparation of piP-HE After digestion of piQ8-HE with ScaI-HindIII, ScaI containing the lipoprotein promoter (lpp) excised from pIN-III 113 ompA-1, the lac operator and the ompA gene
-The HindIII fragment was inserted to create piP-HE.

Reference example 2
(Construction of apoaequorin expression vector piP-His6-HE)
An apoaequorin expression vector piP-His-HE having sequences encoding OmpA and histidine hexamer was constructed as follows.
From the apoaequorin secretion expression vector piP-HE obtained according to the method described in Reference Example 1, piP-H deleted by filling-in the EcoRI on the carboxy terminal side with Klenow fragment
Using EΔ2E, an oligonucleotide that encodes a sequence consisting of 6 histidines (Eco-His6-Hind Linker: 5 'AAT-TCC-CAC-CAT-CAC-CA-T) at the HindIII-EcoRI site of piP-HEΔ2E
-CAC-CAT-GGT 3 '(SEQ ID NO: 3) and Eco-His6-Hind Linker: 5'-AG-CTT-ACC-ATG
-GTG-ATG-GTG-GG 3 '(SEQ ID NO: 4) was inserted to prepare piP-His6-HE.

Reference example 3
(Design and chemical synthesis of a gene encoding apoRLBP)
Since the apoRLBP cDNA has not yet been isolated, the gene encoding ApoRLBP was chemically synthesized by the oligonucleotide assembly method using the PCR procedure.
The gene encoding ApoRLBP (184 amino acid residues) is DNASIS software Ver. Designed using 3.7 (Hitachi Software Engineering). At this time, a preferred codon is not used in E. coli, and 11% in the sequence of 552 nucleotides of apoRLBP.
The restriction enzyme sites were introduced. 20 nucleotide overlapping oligonucleotide (40 mer ×
28, 35 mer × 1) were synthesized on a 50 nmol scale by the phosphoamidate method using a Millipore DNA synthesizer (model Expedite), purified by gel purification method, and vacuum-dried. Gene assembly was performed using the following PCR method (Gene 164, 49-53 (1995)); the dried oligo was resuspended in distilled water at a concentration of about 3.3 μg / μl (250 mM) Combine 1 μl each of oligo solution and mix (0.4 μl) each with 0.25 mM dNTP, 5 units E
It was added to a 40 μl PCR reaction mixture containing xTaq polymerase (Takara Shuzo) and 4 μl of 10 × ExTaq buffer (buffer composition not shown on product insert). PC
As the R program, 55 cycles of 94 ° C.-30 seconds, 50 ° C.-30 seconds, 72 ° C.-60 seconds were performed (Perkin Elmer). Assembly reaction mixture (2.5 μl), outer primer set (3.3 μg); HindIII site underlined 15NL 5 'GGC AAGCTT -CCA-GAA-G
TT-ACT-GCC-AGC-GAA-CGT-GCT-TAC-C 3 '(SEQ ID NO: 5) and 33R with Bam HI site underlined
Used for amplification by L 5 ′ GCC GGATCC- TTA-TAA-TAA-ATC-ACC-ATA-AAA-TGC-ATT-AGC-C 3 ′ (SEQ ID NO: 6) (94 ° C.-30 sec, 50 ° C.- 30 cycles of 30 seconds, 72 ° C.-60 seconds).
The amplified fragment (approximately 550 base pairs) on a 1.2% agarose gel is eluted with 6 M NaI and P
Purification was performed using a CR purification kit (Qiagen). The separated fragment was digested with HindIII and BamHI to obtain a HindIII-BamHI fragment. The HindIII-BamHI fragment of the obtained synthetic apoRLBP gene is
It corresponded to 184 amino acid residues and had 11 restriction enzyme recognition sites in 512 nucleotides (FIG. 1). The thus obtained HindIII-BamHI fragment was pUC9-2 (Gene 30, 247-250 (1
984)) to the HindIII / BamHI site to obtain p92-RLBP. The DNA sequence was determined using an Applied Systems DNA sequencer (models 377 and 310).

Reference example 4
(Expression in E. coli of recombinant apoRLBP with OmpA and no polypeptide rich in basic amino acids)
(1) Construction of piP-RLBP By replacing the HindIII-BamHI fragment of the apoaequorin cDNA in piP-HE obtained in Reference Example 1 with the HindIII-BamHI fragment of p92-RLBP obtained in Reference Example 3. , Om
An apoRLBP expression plasmid piP-RLBP having a pA signal peptide was constructed (FIG. 2). The host used was Escherichia coli BL21 strain (Amersham Bioscience).
(2) Expression of recombinant apoRLP in Escherichia coli Seed culture of the bacterial strain having the expression plasmid piP-RLBP obtained in (1) above was performed in 5 ml of Luria-Bertani medium containing ampicillin (50 μg / ml). For 16 hours at 30 ° C, 500
Added to 80 ml LB medium in a ml Sakaguchi flask. After 16 hours of incubation at 37 ° C,
g, collected by centrifugation for 5 minutes, suspended in 20 ml of 50 mM Tris-HCl (pH 7.6), and sonicated in ice using a Brabson model 250 sonifier. As a result of SDS-PAGE analysis,
The expression of apoRLBP without histidine was confirmed. However, the expressed apoRLBP was present as inclusion bodies in the microbial cells (FIG. 3A, lane 4).

Example 1
(Expression in E. coli of recombinant apoRLBP with OmpA and histidine hexamer)
(1) Construction of piP-His-RLBP Replacing the HindIII-BamHI fragment of the apoaequorin cDNA of piP-His6-HE obtained in Reference Example 2 with the HindIII-BamHI fragment of p92-RLBP obtained in Reference Example 3 As a result, an expression plasmid piP-His-RLBP having an OmpA signal peptide and a histidine hexamer was constructed (FIG. 2A). The host used was E. coli BL21
Stock (Amersham Bioscience).
(2) Expression and Purification of Recombinant ApoRLP in E. coli Seed culture of a bacterial strain having the expression plasmid piP-His-RLBP obtained in (1) above was performed using 5 ml of Luria containing ampicillin (50 μg / ml). -Bertani medium at 30 ° C for 16 hours and added to 80 ml LB medium in a 500 ml Sakaguchi flask. After 16 hours of incubation at 37 ° C, the cells are harvested by centrifugation at 5000 g for 5 minutes, suspended in 20 ml of 50 mM Tris-HCl (pH 7.6), and using a Brabson model 250 sonifier in ice. The sonication was performed.
As a result of SDS-PAGE analysis, it was confirmed that the protein was expressed as a soluble protein using the piP-His-RLBP expression vector (FIG. 3A, lane 3). Next, 12000 g of ultrasonic disruption of cultured cells
The supernatant (20 ml) obtained by centrifugation for 10 minutes was applied to a nickel-chelate column (1.5 × 4 cm) equilibrated with 50 mM Tris-HCl (pH 7.6). Add 50 ml of 50 mM Tris-HCl (pH
In 7.6), the adsorbed protein was eluted with 50 mM Tris-HCl (pH 7.6) containing 0.05 M, 0.1 M, 0.3 M, 0.5 M, and 1 M imidazole by 20 ml each stepwise. The fraction of apoRLBP with histidine tag was eluted with 0.1-0.3 M imidazole, and SDS-P
Analyzed by AGE. ApoRLBP fractions were combined, dialyzed against 4 L of 50 mM ammonium bicarbonate (pH 8.3) and stored at -80 ° C. The yield of purified apoRLBP is 18.2 mg from 80 ml cultured cells
Met.
By SDS-PAGE analysis under heat treatment and reducing conditions, purified apoRLBP showed a purity of 95% or more and a molecular weight of 24 kDa (FIG. 3B, lane 2). This result suggests that the expression level of apoRLBP in E. coli cells is 10% or more of the total protein extracted from bacterial cells. This indicates that the production method of the present invention can produce a large amount of the target protein as a soluble protein.
In the purified apoRLBP, mass spectrometry was performed using MALDI-TOF-MS in order to confirm that the OmpA signal peptide was cleaved correctly and apoRLBP having a histidine hexamer. Mass values of m / z 21931.7 for [M + H] + and m / z 10965.9 for [M + 2H] 2+ were observed. These values were in good agreement with the calculated average mass of 21932.9 for apoRLBP with histidine hexamer without OmpA signal peptide. This result indicates that the OmpA signal peptide of apoRLBP with histidine hexamer was correctly cleaved during the transition from cytoplasm to periplasm in E. coli cells.

Example 2
(Preparation of RLBP from ApoRLBP)
The purified apoRLBP (1 mg = 46 nmol) obtained in Example 1 was added to 10 mM EDTA and 0.66 mM.
It melt | dissolved in 5 ml of 30 mM Tris-HCl (pH7.6) containing DTT, and conversion from apo RLBP to RLBP was started by adding coelenterazine (20 μg = 47 nmol). After standing at 4 ° C. for 16 hours, the mixture was concentrated to 0.5 ml at 4 ° C. using a centrifugal filter (Amicon Ultra, molecular weight cut: 10000) to obtain a yellow-green solution. To remove unbound coelenterazine, the solution was applied to a Sephadex G-25 column (1 x 6 cm) equilibrated with 30 mM Tris-HCl (pH 7.6) containing 10 mM EDTA. Eluted with. 1 fraction (0.5 ml)
Recovered at a flow rate of 0.5 ml per minute and monitored by absorbance at 280 nm and 446 nm. The main fraction of RLBP was eluted from 0.5 ml to 1.0 ml. The protein recovery was 80%. Obtained purified R
The results of SDS-PAGE analysis of LBP under heat treatment and reduction conditions are shown in lane 3 of FIG. 3B.
The purity of the purified RLBP is determined based on the absorption spectrum data of natural RLBP (J. Biol. Chem. 254
, 769-780 (1979)), it was estimated to be over 95%.
This result shows that the apo RLBP obtained in Example 1 binds to coelenterazine to form RLBP, which is a holoprotein, in the same manner as natural RLBP. Thereby, it was confirmed that the recombinant apoRLBP obtained in Example 1 has the original function.

Example 3
(Spectrum analysis of recombinant RLBP)
Absorption spectrum of purified recombinant RLBP is 20 mM containing 2 mM EDTA or 10 mM CaCl ₂
A quartz cuvette (10 mm optical path) at 25 ° C in a Tris-HCl (pH7.6) Jusco (Tokyo) V-560 spectrophotometer (bandpass 0.5 nm; response Quick; scan speed 100 nm / min) And measured. The absorption spectrum of recombinant RLBP in the presence and absence of calcium ions is shown in FIG. Recombinant RLBP has two characteristic absorption maxima at 277 nm and 446 nm, shouldered at 475 nm, which is almost consistent with the spectrum of natural RLBP (J. B
iol. Chem. 254, 769-780 (1979)). The extinction coefficients (ε) of recombinant RLBP at 277 nm and 446 nm were 24900 (E _0.1% = 1.13) and 9300 (E _0.1% = 0.42), respectively. The absorption ratio of recombinant RLBP at 277 nm / 446 nm is 2.68, and the ratio of natural RLBP at 277 nm / 446 nm
Close to 2.7 to 2.8 (J. Biol. Chem. 254, 769-780 (1979)). In the presence of 10 mM calcium ions, the 277 nm and 446 nm peaks of recombinant RLBP were shifted to 265 nm and 434 nm, indicating properties similar to natural RLBP (J. Biol. Chem. 254, 769). -780 (1979)). The absorption peak of coelenterazine at 420 nm (ε = 9300) in the same buffer containing 10 mM EDTA shifted to 432 nm (ε = 10000) in the presence of 10 mM calcium ions. This absorption peak was close to 434 nm of recombinant RLBP in the presence of 10 mM calcium ions.
In addition, fluorescence spectra were measured using a Jusco FP-6500 W fluorescence spectrophotometer (emission and excitation bandpass 5 nm; response 0.5 seconds; scan rate 100 nm / min). The fluorescence spectrum of RLBP excited at 446 nm showed an emission peak at 530 nm with a half bandwidth of 86 nm, and the intensity of the emission peak increased 4 times by the addition of calcium ions.
These spectral change data indicate that the recombinant RLBP has a conformation similar to that of natural RLBP, suggesting that it is normally folded.
Judging from the absence of change in the absorption spectrum, the solution of recombinant RLBP was stable for more than 6 months at 4 ° C and -80 ° C. This indicates that, generally, when a neutral aqueous solution of coelenterazine is stored at 4 ° C., the half-life is about 3 days, but it can be stably stored by using recombinant RLBP.

Example 4
(Luminescence reaction by RLBP and Renilla luciferase)
50 mM Tris-HCl (pH 7.6), 10 mM CaCl ₂ and 5 μg of the recombinant RLBP obtained in Example 3
Add a Renilla luciferase (0.08 μg) to the reaction mixture (100 μl) and use a luminometer (AB2200, manufactured by Ato (Tokyo)) equipped with a photomultiplier tube (R4220P, Hamamatsu Photonics). As a result, continuous light emission was observed (FIG. 5). This shows that coelenterazine contained in the RLBP molecule was released by binding of calcium ions to RLBP, and the released coelenterazine became a substrate of Renilla luciferase and oxidized as a result of catalytic action, resulting in light emission. ing. This result indicates that the RLBP obtained in Example 2 (the combination of apoRLBP obtained in Example 1 and coelenterazine) releases coelenterazine by calcium ions. This resulted in the recombinant apoRLBP obtained in Example 1 and the recombinant RLB obtained in Example 3.
It was confirmed that P has the original function.

Example 5
(Preparation of RLBP from apoRLBP and coelenterazine derivatives)
A novel RLBP was prepared using a coelenterazine derivative in the same manner as in the preparation of RLBP using apoRLBP and coelenterazine described in Example 2. As the coelenterazine derivative, h-coelenterazine, e-coelenterazine, Bis-coelenterazine are used, and the corresponding RL
BP, ie h-RLBP, e-RLBP, Bis-RLBP, was obtained. Apo RLBP,
Calcium ions (Ca ^{2+) of} coelenterazine and RLBP obtained as described above
Table 1 shows the absorption spectrum data in the absence and presence. The absorption spectrum was measured in the same manner as in Example 3 in 50 mM Tris-HCl (pH 7.6) containing 2 mM EDTA or 10 mM CaCl ₂ . Even when the obtained RLBP was stored at 4 ° C. and -80 ° C. for 6 months or more, these absorption spectra showed almost no change. This result indicates that the solution of recombinant RLBP is stable for more than 6 months at 4 ° C and -80 ° C. That is, like coelenterazine, it was shown that coelenterazine derivatives are unstable in solution, but can be stored more stably by using apoRLBP.

Example 6
(Construction of a novel basic vector, piP-H6-M (11), for expressing secreted proteins having a histidine sequence at the amino terminus)
PiP-H, a basic vector for expressing a target protein as a soluble protein
6-M (11) was constructed by the following procedure. Starting vector piP-His6-HE
Was prepared according to the method described in Reference Example 2. Furthermore, a multi-cloning site (NcoI / HindIII / NdeI / SacI) is added to the HindIII-BamHI site of the piP-His6-HE vector.
/ KpnI / XhoI / BamHI / EcoRI / SalI / PstI / XbaI)
iP-H6-M (11) was constructed. The basic vector piP-H6-M (11) is controlled by the E. coli lipoprotein promoter, the lactose operator, for secretion.
OmpA sequence, 6 histidine sequence, multiple cloning site (EcoRI / NcoI /
HindIII / NdeI / SacI / KpnI / XhoI / BamHI / EcoRI / SalI / PstI / XbaI / BamHI
) (FIG. 6).

According to the production of the target protein of the present invention, since the target protein can be produced as a soluble protein, it is not necessary to denature (solubilize) the target protein. Therefore,
According to the present invention, the target protein can be obtained efficiently and at a high recovery rate. Therefore, the present invention is extremely useful as a method for producing useful proteins and proteins such as proteins to be analyzed for functions and structures.
In addition, since the expression vector of the present invention can be produced as a soluble protein by inserting a gene encoding the protein of interest into a restriction enzyme site and expressing it, it can be used for producing a desired protein such as a useful protein. It can be used suitably.

FIG. 1 shows the base sequence and restriction enzyme recognition site of the synthetic apoRLBP gene obtained in Reference Example 3, and the amino acid sequence of apoRLBP. (A) A restriction enzyme map is shown. The shaded area indicates the loop region of the EF hand motif. (B) shows the base sequence of the synthetic apoRLRL gene. A portion surrounded by a square indicates a loop region of the EF hand motif. FIG. 2 shows the expression vector of apoRLBP having OmpA signal peptide and histidine hexamer obtained in Example 1 piP-His-RLBP and the expression vector of apoRLBP having OmpA signal peptide obtained in Reference Example 4. piP-RLBP is shown. (A) shows a restriction map of piP-His-RLBP. (B) shows the amino terminal amino acid sequence of piP-His-RLBP and piP-RLBP, and the cleavage site of the signal peptide sequence. FIG. 3 shows the results of SDS-PAGE analysis of the recombinant apoRLBP obtained in Example 1, the recombinant RLBP obtained in Example 2, and the recombinant apoRLBP obtained in Reference Example 4. Samples in each lane are as follows. (A) Lane 1: Protein molecular weight marker (Tefco): β-galactosidase (116,000), phospholipase B (97,400), bovine serum albumin (69,000), glutamate dehydrogenase (55,000), lactate dehydrogenase (36,500), carbonic acid dehydrogenase (29,000), trypsin inhibitor (20,100); lane 2: precipitate obtained by centrifuging an ultrasonic disruption of cultured cell piP-His-RLBP / BL21 (40 μl) in Example 1; lane 3: culture in Example 1 Supernatant obtained by centrifuging ultrasonic disruption of cell piP-His-RLBP / BL21 (40 μl); Lane 4: sonication disruption of cultured cell piP-RLBP / BL21 (40 μl) in Reference Example 4 Obtained precipitate; Lane 5: Over culture cell piP-RLBP / BL21 (40 μl) in Reference Example 4 Supernatant obtained by the waves breaking was centrifuged. (B) Lane 1: protein molecular weight marker; Lane 2: purified apoRLBP (2.2 μg) bound to histidine hexamer obtained in Example 1; Lane 3: bound to histidine hexamer obtained in Example 2 Purified RLBP (3.5 μg). FIG. 4 shows the absorption spectrum of recombinant RLBP in Example 3 in the absence and presence of calcium ions. A solid line and a broken line show absorption spectra in the absence and presence of calcium ions, respectively. The protein concentration was 0.45 mg / ml. FIG. 5 shows the luminescence reaction by recombinant RLBP obtained in Example 3 and Renilla luciferase in Example 4. The inset shows an enlarged view of the part indicated by the arrow. FIG. 6 shows the expression vector piP-H6-M (11) obtained in Example 6.

[SEQ ID NO: 1] This shows the amino acid sequence of apoRLBP.
[SEQ ID NO: 2] This shows the base sequence of synthetic DNA encoding apoRLBP.
[SEQ ID NO: 3] This shows the base sequence of the oligonucleotide encoding the sequence consisting of 6 histidines used in Reference Example 2 and Example 6.
[SEQ ID NO: 4] This shows the base sequence of the oligonucleotide encoding the sequence consisting of 6 histidines used in Reference Example 2 and Example 6.
[SEQ ID NO: 5] This shows the base sequence of the primer used in Reference Example 3.
[SEQ ID NO: 6] This shows the base sequence of the primer used in Reference Example 3.

Claims (9)

Expressing a protein using a polynucleotide encoding a secretion signal peptide, a polynucleotide encoding a polypeptide rich in basic amino acids, and a polynucleotide encoding a target protein in this order,
The secretion signal peptide is a secretion signal peptide (OmpA) derived from the outer membrane protein A of E. coli,
A polypeptide rich in basic amino acids is polyhistidine,
Target protein apoaequorin, Apokuraichiin, der any one selected from the group consisting of apoobelin and apo apomitrocomin is,
Method for producing the target protein as a soluble protein expression of the protein is characterized Rukoto place within Gram-negative bacteria is a host cell. The method according to claim 1 , wherein the gram-negative bacterium is a bacterium of the genus Escherichia coli. The method according to claim 1 or 2 , wherein the polypeptide rich in basic amino acids is a polypeptide consisting of 5 to 12 amino acid residues. The method according to any one of claims 1 to 3 , wherein the target protein is expressed using an expression vector containing a polynucleotide encoding the target protein. A polynucleotide comprising, in this order, a polynucleotide encoding a secretion signal peptide of a Gram-negative bacterium, a polynucleotide encoding a polypeptide consisting of 5 to 12 basic amino acid residues, and a polynucleotide encoding a target protein Using nucleotides to express proteins in gram-negative bacteria,
The secretion signal peptide of Gram-negative bacteria is a secretion signal peptide (OmpA) derived from the outer membrane protein A of E. coli,
A polypeptide consisting of 5 to 12 basic amino acid residues is polyhistidine,
A method for producing the target protein as a soluble protein, wherein the target protein is any one selected from the group consisting of apoaequorin, apocritin, apoovelin, and apomytrocomin. Expressing a protein in a bacterium of the genus Escherichia coli using a polynucleotide that contains a polynucleotide encoding OmpA, a polynucleotide encoding polyhistidine, and a polynucleotide encoding a target protein in this order ;
A method for producing the target protein as a soluble protein, wherein the target protein is any one selected from the group consisting of apoaequorin, apocritin, apoovelin, and apomytrocomin. (A) a first coding region encoding a secretory signal peptide;
(B) a second coding region encoding a polypeptide rich in basic amino acids, and (c) a third coding region encoding a target protein.
In this order ,
The secretion signal peptide is a secretion signal peptide (OmpA) derived from the outer membrane protein A of E. coli,
A polypeptide rich in basic amino acids is polyhistidine,
An expression vector, wherein the target protein is any one selected from the group consisting of apoaequorin, apocritin, apoovelin and apomytrocomin. The expression vector according to claim 7 , wherein the polypeptide rich in basic amino acids is a polypeptide comprising 5 to 12 amino acid residues. (A) a first coding region encoding a secretory signal peptide derived from a Gram-negative bacterium,
(B) a second coding region encoding a polypeptide consisting of 5 to 12 basic amino acid residues, and (c) a third coding region encoding a target protein.
In this order ,
The secretion signal peptide derived from Gram-negative bacteria is a secretion signal peptide (OmpA) derived from the outer membrane protein A of E. coli,
A polypeptide rich in 5 to 12 basic amino acids is polyhistidine,
An expression vector, wherein the target protein is any one selected from the group consisting of apoaequorin, apocritin, apoovelin and apomytrocomin.

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