JP2008220240A - METHOD FOR PRODUCING HUMAN TYPE CYTOCHROME c AND PROTEIN DERIVED FROM EUKARYOTE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for mass-producing a protein (recombinant protein) holding the structure and function of natural protein derived from a eukaryote, particularly a hemoprotein such as human type cytochrome c using a genetic engineering technique, to provide a gene cassette useful for producing the protein by the genetic engineering technique, and to provide an expression vector comprising the gene cassette. <P>SOLUTION: The method for production comprises a step of culturing a bacterium transformed with a vector containing a gene encoding a signal peptide composed of an amino acid sequence described in sequence number 1 and a gene encoding the protein derived from the eukaryote and belonging to the genus Shewanella and collecting the protein derived from the eukaryote and produced in the periplasma of the bacterium from the resultant culture product. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for producing a large amount of a protein (recombinant protein) retaining the structure and function of a natural protein derived from a eukaryote using genetic engineering techniques. In particular, the present invention relates to a method for producing a large amount of a heme protein such as cytochrome c derived from human using genetic engineering techniques. The present invention also relates to a gene cassette useful for producing the protein derived from the eukaryote by genetic engineering techniques and an expression vector containing the gene cassette.

  Cytochrome c is an electron transport enzyme (soluble protein) that controls the redox reaction in cellular respiration. Recently, involvement in apoptosis has been pointed out, but various symptoms caused by tissue oxygen deficiency such as cerebral blood flow, cerebrovascular disorders such as cerebral softening, carbon monoxide poisoning, dyspnea due to lung disease, etc. It is known to be effective in improving the blood pressure, and is actually used clinically as a tissue respiratory activator (for example, Non-Patent Document 1). However, there is no conventional industrial production technology of human cytochrome c, and the above pharmaceuticals are also made from equine myocardium-derived cytochrome c as a raw material due to restrictions on the supply source. Since equine cytochrome c has 12 amino acid residue differences from human cytochrome c, a pharmaceutical product derived from equine cytochrome c may be antigenic to humans. For example, precautions regarding the use of the above tissue respiratory activator include shock symptoms as hypersensitivity, sometimes facial edema, rash, measles, etc., gastrointestinal symptoms such as nausea, abdominal pain, diarrhea, It is known that symptoms such as headache, numbness, head feeling, and anxiety appear in the nervous system (Non-patent Document 1).

  In addition, the price of cytochrome c sold as a reagent is about 60,000 yen per gram of bovine cytochrome c, but it is currently very high at 70 to 90,000 yen per 10 μg of human cytochrome c. is there.

  For this reason, development of a method for industrially producing human cytochrome c by genetic engineering techniques is required. For this purpose, as a method for synthesizing human cytochrome c by a genetic recombination method, a method using yeast has been reported, but there is a problem that the production efficiency is low (Patent Document 1). Therefore, although it is desired to synthesize cytochrome c in a prokaryotic organism having a high production capacity, for example, E. coli, there is no report that cytochrome c has been synthesized efficiently in E. coli at present.

In Patent Document 2, a vector in which a DNA encoding a signal peptide derived from red algae, particularly Susavinori, is bound to the 5 ′ end of a DNA encoding cytochrome c is used for prokaryotes such as Escherichia coli. Thus, a method for producing eukaryotic cytochrome c including mammals is described. However, its production efficiency is not necessarily high, and even with the recombinant cytochrome c derived from Susabiori, the yield is only about 2 mg per 10 liters of culture.
Tissue respirator “Citrest” (Mochida Pharmaceutical Co., Ltd.) package insert JP-A 64-37291 JP 2002-218879 A

  An object of the present invention is to provide a method for producing a protein derived from a eukaryote such as an animal or plant as an active protein having its structure and function by using a gene recombination method. Specifically, the present invention relates to proteins having covalently bound heme such as cytochrome c, proteins having a disulfide bond between molecules or between molecules such as protein kinases, etc. in a state in which the structure, function and activity are retained. The object is to provide a method for expression production. Furthermore, an object of the present invention is to provide a gene cassette that can be effectively used for the production of foreign proteins such as the above-mentioned heme protein using genetic engineering techniques, and an expression vector containing the gene cassette.

  In view of the above-mentioned conventional problems, the present inventors have studied to develop a method for easily and in large quantities producing a protein derived from a eukaryote, particularly human cytochrome c, which is a heme protein, by a gene recombination method. Has been repeated. As a result, by using a gene encoding a specific signal peptide derived from a microorganism belonging to the genus Schwannella as a secretory signal gene, an active cytochrome c in which heme is normally bound in the periplasm of the bacterium belonging to the genus Schwannella. Was successfully produced in large quantities. In Escherichia coli, the enzyme for binding heme to protein is deficient, whereas the genus Shuwanella has periplasm and has its enzyme group constantly. Therefore, transporting heme to the periplasm is the first condition for producing active cytochrome c. In other words, the fact that active cytochrome c could be produced means that the signal peptide functions effectively to transfer the precursor of cytochrome c before heme binding to the periplasm.

  In addition, the present inventors similarly maintain the normal activity of protein kinase 2 derived from eukaryotes in the periplasm of bacteria belonging to the genus Shuwanella by using a gene encoding the specific signal peptide. We succeeded in producing in the state. In general, protein expression in the periplasm of Gram-negative bacteria such as Escherichia coli is less affected by proteases than in cytoplasm, and the risk of digestion of the produced protein is low. Furthermore, because the periplasm is a reducing environment, unlike the cytoplasm, there is no possibility of creating wrong intramolecular or intermolecular disulfide bonds. Therefore, the fact that a protein kinase having normal activity could be produced by the method of the present invention means that the protein is transferred to the periplasm of bacteria belonging to the genus Schwanella by the action of the signal peptide, thereby escaping from digestion of protease. This is considered to mean that the formation of an erroneous disulfide bond was suppressed.

  Based on these facts, the present inventors use a gene encoding a Schwannella signal peptide as a signal gene and a bacterium belonging to the genus Schwannella as a host for eukaryotic proteins such as heme proteins and protein kinases. In order to complete the present invention, it was confirmed that it could be produced easily and in good yield in terms of genetic engineering while maintaining the structure such as the heme bond structure and disulfide bond structure, and the function and activity. It came.

The present invention is based on such findings, and specifically includes the following embodiments:
(I) Method for producing eukaryotic protein
I-1. Culturing a bacterium belonging to the genus Shuwanella transformed with a vector containing a gene encoding a signal peptide consisting of the amino acid sequence described in SEQ ID NO: 1 and a gene encoding a protein derived from a eukaryote, and the resulting culture A method for producing a eukaryotic protein, comprising a step of collecting a eukaryotic protein produced in the periplasm of the bacterium from a product.
I-2. Production according to I-1 wherein the gene encoding the signal peptide has the base sequence shown in SEQ ID NO: 2 or has a base sequence in which one to several bases are substituted in the base sequence Method.
I-3. The production method according to I-1 or I-2, wherein the eukaryotic protein is a heme protein or a protein having a disulfide bond within or between molecules.
I-4. The production method according to I-3, wherein the heme protein is human cytochrome c.
I-5. The production method according to any one of I-1 to I-4, which comprises a step of transforming a bacterium belonging to the genus Shuwanella using a foreign protein expression vector for the bacterium belonging to the genus Shuwanella described in III-1 described later.

(II) Gene cassette for foreign protein expression
II-1. A gene cassette for expressing a foreign protein used for producing a foreign protein in the periplasm of a bacterium belonging to the genus Shuwanella, which has any one of the following base sequences (1) to (3):
(1) (a) a promoter that is expressed in a bacterium belonging to the genus Shuwanella, and a base sequence comprising a gene encoding a signal peptide consisting of the amino acid sequence described in SEQ ID NO: 1 linked downstream thereof, (b)
(2) (b) The 3 'end of the gene encoding the signal peptide consisting of the amino acid sequence described in SEQ ID NO: 1 and (c) the 5' end of the foreign protein structural gene so that the amino acid frame is continuous. A base sequence formed by ligation,
(3) (a) a promoter expressed in a bacterium belonging to the genus Shuwanella, and (b) 3 ′ end of a gene encoding a signal peptide consisting of the amino acid sequence described in SEQ ID NO: 1 linked downstream thereof, ( c) A nucleotide sequence in which the 5 'end of a foreign protein structural gene is linked so that the amino acid frame is continuous.

II-2. A gene cassette for expressing a foreign protein according to II-1, wherein the gene cassette has the base sequence of (2) above and is used by being linked downstream of a promoter expressed in a bacterium belonging to the genus Shuwanella .
II-3. II-1 or II-2, wherein the gene encoding the signal peptide has the base sequence described in SEQ ID NO: 2 or has a base sequence in which 1 to several bases are substituted in the base sequence The gene cassette described in 1.
II-4. The gene cassette according to any one of II-1 to II-3, wherein the foreign protein is a heme protein or a protein having a disulfide bond within or between molecules.
II-5. The gene cassette described in II-4, wherein the heme protein is human cytochrome c.

(III) Foreign protein expression vector
III-1. A foreign protein expression vector for bacteria belonging to the genus Shuwanella, comprising the gene cassette for expressing a foreign protein according to any one of II-1 to II-5.

(IV) Transformant
IV-1. A bacterium belonging to the genus Shuwanella transformed with a foreign protein expression vector described in III-1.

(V) Foreign protein production method
V-1. Transform bacteria belonging to the genus Shuwanella using the foreign protein expression vector described in III-1, then culture the transformed bacteria, and the foreign material produced in the periplasm of the bacteria from the obtained culture A method for producing a foreign protein, comprising a step of collecting a protein.

  In the present invention, by using a gene encoding a signal peptide derived from a microorganism belonging to the genus Shuwanella described in SEQ ID NO: 2 as a secretory signal gene, human cytochrome c or the like is contained in the periplasm of a bacterium belonging to the genus Shuwanella. It has become possible to produce proteins derived from eukaryotes, such as activated heme protein, to which heme is normally bound, in a yield with the natural structure and function retained. That is, by using the technology provided by the present invention, it is difficult to produce a heme protein (particularly, human cytochrome c), which has been difficult to produce in a state in which the conventional gene recombination method retains its natural structure and activity. Can be manufactured in large quantities.

  In Escherichia coli, the enzyme for binding heme to protein is deficient, whereas the genus Shuwanella has periplasm and has its enzyme group constantly. Therefore, transporting heme to the periplasm is the first condition for producing active cytochrome c. In addition, protein expression in the periplasm of the genus Shuwanella is less affected by proteases than in the cytoplasm, so even proteins that do not appear to be expressed in E. coli can be obtained normally in the expression system. It is. In addition, conventional expression in E. coli is expressed in the cytoplasm and may create wrong intramolecular or intermolecular disulfide bonds, whereas the periplasm is in a reducing environment, so normal disulfide bonds Can be made. In addition, since the signal peptide provided by the present invention is automatically cleaved by the host after protein expression, the target protein can be obtained as a mature form. Furthermore, proteins expressed in the periplasm can be obtained by extracting the periplasmic fraction (Davidson, VL and Sun, D. (2002) Lysozyme-Osmotic Shock Methods for Localization of Periplasmic Redox Proteins in Bacteria. Meth. Enzymol. Vol. 353, pp. 121-130.), It is expected that there will be less contamination of host-derived proteins and the purification process can be reduced.

  Moreover, according to the gene cassette provided by the present invention, the protein production method according to the above method can be easily carried out.

(I) Method for Producing Eukaryotic Protein The method for producing a eukaryotic protein of the present invention comprises a gene encoding a signal peptide derived from the genus Shuwanella and a gene encoding a target protein derived from a eukaryote. Culturing a bacterium belonging to the genus Shuwanella, transformed with an expression vector containing the protein in a state that can be expressed in the bacterium belonging to the genus Shuwanella (host), and collecting the protein produced in the periplasm of the bacterium from the culture It is characterized by doing.

  The proteins targeted by the present invention are derived from eukaryotes (animals such as vertebrates including humans and invertebrates including insects; plants; fungi including mushrooms, molds and yeasts; protists including algae). If it is a thing, there will be no restriction | limiting in particular. Examples of eukaryotes include animals such as vertebrates including humans and invertebrates including insects; plants; fungi including mushrooms, molds and yeasts; and protists including algae, but preferably animals. And vertebrates including plants, more preferably humans.

  Specific examples of the protein include a heme protein that has conventionally been difficult to produce by genetic recombination methods using prokaryotic cells such as E. coli and eukaryotic cells such as yeast. Heme proteins are proteins that have iron porphyrin complexes (heme) in their molecules. These hemoproteins include, for example, hemoglobin and myoglobin involved in the transport and storage of enzyme molecules, and peroxidase, catalase, Cytochrome p-450, cytochromes involved in electron transfer, etc. are included. Cytochromes are preferred. The cytochromes include cytochrome a (formyl porphyrin iron) such as cytochrome a1 and cytochrome a3; cytochrome b (prototoporphyrin iron) such as cytochrome b2, cytochrome b5, cytochrome b559 and cytochrome b563, depending on the type of heme contained; Cytochrome c (mesoporphyrin derivative iron) such as c1, c2, c6, c551 and c553 and cytochrome f; and cytochrome d (dihydroporphyrin derivative iron). Cytochrome c derived from eukaryotes is preferable. Examples of the cytochrome c include an electron transport enzyme (membrane protein) present in the inner mitochondrial membrane of animals and plants.

  More preferably, the target protein includes cytochrome c (human cytochrome c) present in vertebrates, particularly human mitochondria. Human cytochrome c is a basic protein consisting of 104 amino acids and having a molecular weight of about 13,000, and its amino acid sequence (SEQ ID NO: 3) and base sequence (SEQ ID NO: 4) are already known (Matsubara, H. et al , J. Biol. Chem. 238, 2732, 1963) (GeneID: 54205).

  The protein of interest is not limited to this, and other proteins such as protein kinases that are affected by proteases in expression in the cytoplasm, or proteins having a disulfide bond within or between molecules.

  The genes (DNA) encoding these proteins can be chemically synthesized based on their known base sequences, or can be obtained from the cDNA library using a nucleic acid probe and by the PCR method. Can do. The cDNA library was prepared by preparing poly (A) RNA from total RNA prepared from the target eukaryote using an oligo (dT) cellulose column according to a conventional method, and then obtaining the poly (A) It can be prepared by making cDNA from RNA and transforming host cells (eg, E. coli) with it.

  The gene encoding the amino acid sequence of the eukaryotic protein may have a base sequence derived from the eukaryote, but any host encoding the amino acid sequence can be used for genetic recombination. It may be designed using a codon that is frequently used in a bacterium belonging to the genus Shuwanella. For example, those listed in Table 1 are known as possible codon types encoding each amino acid.

  The types of codons (amino acid translation codons) that are frequently used depending on the type of cells used as a host have been clarified to some extent, and for example, Gunsum et al., Nuc Acids Res., Vol. 8, pages 1893-1912 1980; Haas et al., Curr. Biol., 6, 315-324, 1996; Wayne-Hobson et al., Gene 13, 355-364, 1981; Gross Jean and Fierce, Gene , 18, 199-209, 1982; Holm, Nuc Acids Res., 14, 3075-3087, 1986; Ikemura, J. Mol. Biol., 158, 573-597, 1982 can be mentioned.

  For example, the natural gene encoding the amino acid sequence of human cytochrome c (SEQ ID NO: 3) has the base sequence shown in SEQ ID NO: 4 as described above, but is not limited to this, for example, the base sequence shown in SEQ ID NO: 5 You may have. Such a base sequence is a base sequence encoding an amino acid sequence of human cytochrome c artificially designed based on Table 2 according to the codon usage frequency of a host (microorganism belonging to the genus Schwanella) used for genetic recombination. . FIG. 3 shows a comparison between the naturally occurring base sequence encoding human cytochrome c (upper) and the artificially designed base sequence (lower), and the ratio of both is 93.02% (293/315). It has the same.

  The expression vector used in the present invention can express a gene encoding a signal peptide derived from the genus Schwannella in addition to the gene encoding the target eukaryotic protein, in a bacterium belonging to the genus Schwannella used as a host. It is characterized by including in a state.

  In general, an expression vector is expressed as necessary in the order of the downstream direction of transcription in order to express a target protein in a target host (1) a promoter functional region, (2) a liposome binding site, (3) a translation initiation codon, (4) DNA having a base sequence encoding a signal peptide, (5) DNA having a base sequence encoding a target structural protein, (6) translation termination codon, (7) terminator, (8) selectable marker gene, (9 ) It is constructed so as to contain an autonomously replicating sequence (replicon) that can self-replicate in the host, (10) a homologous region, and the like.

  In the expression vector used in the production method of the present invention, a protein derived from a target eukaryote is expressed in a bacterium belonging to the genus Shuwanella, and the expressed protein is transferred from the cytoplasm of the cell to the periplasm. It is required to contain a minimum of components for the purpose.

  Such an expression vector includes, among the above components, (1) a promoter functioning as a `` promoter functional region '', a promoter that functions in bacteria belonging to the genus Shuwanella, (4) a `` DNA having a base sequence encoding a signal peptide '', SEQ ID NO: DNA having a base sequence encoding a signal peptide consisting of the amino acid sequence described in 1 is referred to as (5) “DNA having a base sequence encoding a structural protein”, and the base sequence encoding the eukaryotic protein described above is It has a replicon that functions as a (9) “autonomous replication sequence (replicon) capable of self-replication in a host” in bacteria belonging to the genus Shuwanella.

  In the expression vector, (4) “DNA having a base sequence encoding a signal peptide” is identical to (5) “DNA having a base sequence encoding a structural protein” on the 5 ′ end side. It is arranged in such a way that it can be expressed in the downstream region of (1) promoter functional region (promoter that functions in bacteria belonging to the genus Shuwanella), and other components also function. It exists connected in a possible state. In addition, expression possible here means that, when an expression vector is introduced into a bacterium belonging to the genus Shuwanella, the expression vector functions so as to express a gene encoding a target protein in the bacterium. In addition, functioning means that when an expression vector is introduced into a bacterium belonging to the genus Shuwanella, the expression vector functions to express a gene encoding the target protein in the bacterium and produce the target protein. Means.

(4) The signal peptide used in the present invention as “DNA having a base sequence encoding a signal peptide” is characterized by comprising the amino acid sequence set forth in SEQ ID NO: 1. As shown in Example 1 (1), the signal peptide is a signal peptide of a 4-heme protein derived from Shewanella oneidensis MR-1 (GenBank accession number NC_004347) belonging to the genus Shuwanella.

FIG. 1 shows the base sequence (SEQ ID NO: 6) of a gene encoding 4 heme protein derived from the Shewanella oneidensis MR-1 (GeneID: 1170426, locus tag: SO2727). The underlined region (SEQ ID NO: 2) located in front of the coding region corresponds to the region (signal sequence) encoding the signal peptide. The base sequence of the gene encoding the signal peptide is not limited to the base sequence (SEQ ID NO: 2) shown in FIG. 1 as long as it encodes the amino acid sequence shown in SEQ ID NO: 1. One to several bases may be substituted. The substitution can be appropriately selected according to the codon degeneracy shown in Table 1.

  Such a signal sequence can be prepared by chemically synthesizing based on the base sequence shown in the present invention. Techniques for substituting one or a plurality of bases in a known base sequence are known in the art. Examples of such methods include site-directed mutation (Site-directed Mutagenesis), restriction enzyme treatment, synthetic gene methods, The usual genetic engineering techniques such as PCR may be mentioned.

The microorganism used as a host in the present invention is a bacterium belonging to the genus Shewanella having a void called a periplasm between a cell membrane and a cell wall. As bacteria belonging to Shewanella genus, specifically Shewanella abalonesis, Shewanella affinis, Shewanellaalgae, Shewanella amazonensis, Shewanella aquimarina, Shewanella arctica, Shewanella baltica, Shewanellabenthica, Shewanella colwelliana, Shewanella decolorations, Shewanella denitrificanis, Shewanella fidelis, Shewanellafrigidimarina, Shewanella gaetbuli, Shewanella gelidimarina, Shewanella hafniensis, Shewanella halifaxensis, Shewanellahanedai, Shewanella japonica, Shewanella kaireiae, Shewanella livingstonesis, Shewanella loihica, Shewanellamarinintestina, Shewanella marisflavi, Shewanella massilia, Shewanella morhuae, Shewanella olleyana, Shewanellaoneidensis, Shewanella pacifica, Shewanella pealeana, Shewanella piezotolerans, Shewanella pneumatophori , Shewanella profunda , Shewanella putrefaciens , Shewanellasaccharophilus , Shewanella sairae , Shewanella schlegeliana , Shewanella sedimentalis , Shewanella sediminis , Shewanel lasurugaensis , Shewanella taiwanensis , Shewanella violacea , Shewanella waksmanii , Shewanella woodyi , and various Shewanellasp .

Preferred examples include Shewanella frigidimarina , Shewanellaputrefaciens , Shewanella japonica , and Shewanella oneidensis . More preferable examples include Shewanellaoneidensis, especially Shewanella oneidensis MR-1.

  The promoter used in the expression vector of the present invention is not particularly limited as long as it exhibits a promoter function in bacteria belonging to the genus Shuwanella. Preferably, it is derived from Gram-negative bacteria, and specific examples thereof include, but are not limited to, the lac promoter, tac promoter, trp promoter, and the like. More preferably, it is derived from a bacterium belonging to the genus Shuwanella.

  These promoters may be partly modified or modified as long as they exhibit the promoter function (promoter transcription activity) in the expression system.

  The replicon used in the expression vector of the present invention may be any replicon that functions so as to be capable of self-replication in bacteria belonging to the genus Shuwanella. Such a replicon may be derived from a bacterium belonging to the genus Shuwanella, but as shown in the examples described later, it does not necessarily have to be derived from a bacterium belonging to the genus Shuwanella used as a host. It is also possible to use replicons derived from gram-negative bacteria.

  The expression vector of the present invention may have any replicon that functions in bacteria belonging to the genus Shuwanella as an autonomously replicating sequence that can self-replicate in the host. A replicon that does not function in bacteria). Such a replicon is not particularly limited, and examples thereof include replicons derived from algae such as E. coli, yeast, Bacillus subtilis, radiobacterium, cyanobacteria, plant cells, insect cells, and mammalian cells. By having a replicon that functions in bacteria belonging to the genus Schwanella and other replicons as autonomously replicating sequences, the expression vector can be expressed in the cells of the bacteria belonging to the genus Schwanella and other organisms (for example, in the biological cells derived from the replicon). ) And can be positioned as a shuttle vector in that sense. For example, an expression vector containing two replicons that function in bacteria belonging to the genus Schwanella and a replicon derived from Escherichia coli is a shuttle vector constructed in a state in which they can replicate with each other between the bacteria belonging to the genus Schwanella and E. coli. be able to.

  Such an expression vector of the present invention may further contain at least one selectable marker gene for convenience of manipulation.

As the selection marker gene, an antibiotic resistance gene or an auxotrophic gene is generally exemplified. Specifically, when the host used for expression of the gene encoding the target protein is a bacterium as in the present invention, an antibiotic resistance gene can be used, and the antibiotic resistance gene includes a cycloheximide resistance gene, Examples include ampicillin resistance gene (β-lactamase gene), tetramycin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene, bleomycin resistance gene, hygromycin resistance gene, G-418 resistance gene and the like. In addition, these selection markers can be used 1 type or in combination of 2 or more types. However, it has been clarified that ampicillin resistance gene and rifampicin resistance gene exist in the host of Shewanella oneidensis MR-1, so when using the bacterium as a host, use antibiotic resistance genes other than these two. Is preferred. Preferably, a kanamycin resistance gene and a tetracycline resistance gene are preferable.

  The expression vector of the present invention comprises, as necessary, other components (2) liposome binding site, (3) translation initiation codon, (6) translation termination codon, (7) terminator, (10) homology region, etc. One or a combination of two or more can be included. These components (DNA sequences) are preferably linked so that when the expression vector is introduced into the host, the gene encoding the target protein functions so that it can be expressed and produced in the host.

  Here, for example, ATG is exemplified as a translation initiation codon, and TAA, TGA, or TAG is exemplified as a translation termination codon. Each of these codons may be arranged in combination of one or more in each region as necessary, and there is no particular limitation thereto.

  The terminator is not particularly limited as long as it corresponds to a host used for expression of a gene encoding a target protein, and can be selected based on common technical knowledge in the art.

  In addition, the homologous region for introduction into the host chromosome is not particularly limited as long as it corresponds to the host used for expression of the gene encoding the target protein, and is based on common general technical knowledge in the art. You can choose.

  Such an expression vector can be prepared by inserting each functional component (DNA component) into a known plasmid. Each functional component can be inserted into a plasmid according to a general method of DNA recombination, for example, the method described in Molecular Cloning. (1989), (Cold Spring Harbor Lab.).

  The expression vector described above is an expression vector that is preferably used in the production method of the present invention. In the present invention, instead of this, an expression vector containing a gene cassette for expressing a foreign protein described later is used. You can also.

  The production method of the present invention includes a step of preparing a transformant by introducing the expression vector thus prepared into a bacterium belonging to the genus Shuwanella.

The transformant can be prepared by introducing the above-described expression vector into a bacterium belonging to the genus Shuwanella as a host by a known method. For the introduction method, a conventional method used for introducing an expression vector into E. coli can be used in the same manner. Specifically, the competent cell method (J. Mol. Biol., 53 , 154, 1970) can be used. ), Electric pulse method (Proc. Natl. Acad. Sci. USA, 81 , 7161, 1984), calcium chloride method, rubidium chloride method and the like can be exemplified without limitation.

The host used in the present invention is a bacterium belonging to the genus Shuwanella. Specific examples of the bacterium belonging to the genus Shuwanella can include those described above. Preferably, Shewanella frigidimarina, Shewanella putrefaciens, Shewanellajaponica , a Shewanella oneidensis, more preferably Shewanella oneidensis, a inter alia Shewanella oneidensis MR-1.

  The expression vector in the host cell is inserted into the chromosome or replaced. Alternatively, it may exist in a plasmid state. The copy number of the foreign gene introduced into the host may be one copy or multiple copies.

  The transformant thus obtained can then be cultured in an appropriate medium depending on the host in order to produce the target protein. The transformant described above is also an object of the present invention, and the present invention also provides such a transformant.

The medium contains a carbon source, a nitrogen source, an inorganic substance, a vitamin, a drug and the like essential for the growth of the transformant. Examples of the medium include LB medium (Nissui Pharmaceutical), chemical synthesis medium (Takayama, Y., Shen, Y., and Akutsu, H. (2007) J. Biochem . 141, 121-126), 2xYT medium ( Molecular Cloning), CHL medium (Takayama, Y., Kobayashi, Y., Yahata, N., Saitoh, T., Hori, H., Ikegami, T., and Akutsu, H. (2006) Biochemistry 45, 3163- 3169). A 2 × YT medium is preferable.

  The culture is usually carried out in the temperature range of 4 to 30 ° C. for about several to 360 hours, and aeration and agitation can be added as necessary. The culture temperature is preferably 25 to 30 ° C.

  The production method of the present invention is further carried out by collecting a target eukaryote-derived protein from the culture thus obtained. The target protein can be collected by extracting the target protein accumulated in the periplasm of the transformant (bacteria belonging to the genus Shuwanella) by a known method after the culture, and purifying it as necessary. . At this time, the host bacteria obtained by culturing may be disrupted, whereby the target protein accumulated in the periplasm can be taken out into the culture supernatant. Separation and purification are, for example, known methods such as salting out, solvent precipitation, dialysis, ultrafiltration, gel electrophoresis, gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, affinity chromatography, etc. A combination of techniques can be performed.

(II) Gene cassette for expressing a foreign protein The present invention provides a gene cassette for expressing a foreign protein that is useful for simply producing the eukaryotic protein described above.

  In the present invention, the “gene cassette for expressing a foreign protein” is a set of DNAs that are incorporated into a “foreign protein expression vector” and used to express a desired foreign protein in the periplasm of a cell belonging to the genus Shuwanella. . The “foreign protein expression vector” targeted by the present invention is used by being introduced into a cell belonging to the genus Shuwanella used as a host, and is a DNA necessary for expressing the foreign protein when introduced. Consisting of a set of The vector is a linear or circular structure that can be replicated in cells belonging to the genus Shuwanella, and includes the above-mentioned “gene cassette for expressing foreign protein”.

Examples of DNA that the foreign protein expression cassette has as a set include the following combinations of two or more of (a) to (c):
(a) Promoter expressed in bacteria belonging to the genus Shuwanella
(b) a gene encoding a signal peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1,
(c) Foreign protein structural gene.

Examples of these combinations include (a) and (b), (b) and (c), and (a), (b), and (c). (1) to (3) are included in any of the embodiments:
(1) (a) a promoter expressed in a bacterium belonging to the genus Shuwanella, and (b) a gene encoding a signal peptide consisting of the amino acid sequence described in SEQ ID NO: 1 linked downstream thereof,
(2) (b) The 3 'end of the gene encoding the signal peptide consisting of the amino acid sequence described in SEQ ID NO: 1 and (c) the 5' end of the foreign protein structural gene so that the amino acid frame is continuous. An aspect formed by linking,
(3) (a) a promoter expressed in a bacterium belonging to the genus Shuwanella, and (b) linked to the downstream thereof (b) at the 3 ′ end of a gene encoding a signal peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, c) An embodiment in which the 5 ′ end of the foreign protein structural gene is linked so that the amino acid frame is continuous.

  The protein expression cassette of the present invention comprising the aspect of (3) is further optionally (i) a liposome binding site, (ii) a translation initiation codon, (iii) a translation termination codon, (iv) a terminator, (v) A selection marker gene, (vi) an autonomously replicating sequence (replicon) capable of self-replication in a host, (vii) one or more homologous regions may be included. Moreover, the protein expression cassette of the present invention comprising the aspect of (1) comprises (c) a DNA encoding a foreign protein structural gene in addition to one or more arbitrary DNAs comprising the above (i) to (vii). In addition, the protein expression cassette of the present invention according to the embodiment (2) includes, in addition to one or more arbitrary DNAs comprising (i) to (vii), (a) a promoter expressed in a bacterium belonging to the genus Shuvanella May be included.

  In addition, in order to express the target foreign protein in the introduced host (bacteria belonging to the genus Shuwanella), these DNAs are expressed in the order of downstream transcription (a) in the bacteria belonging to the genus Schwannella. A promoter, (i) a liposome binding site, (ii) a translation initiation codon, (b) a gene encoding a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, (c) a foreign protein structural gene, (iii) a translation termination codon It is desirable that (iv) a terminator, (v) a selectable marker gene, (vi) an autonomously replicating sequence (replicon) capable of self-replication in a host, and (vii) a homologous region.

  Here, “(a) promoters expressed in bacteria belonging to the genus Shuwanella” can be widely used those that exhibit a promoter function in bacteria belonging to the genus Shuwanella. Preferably, it is derived from Gram-negative bacteria, and specific examples thereof include lac promoter, tac promoter and trp promoter. More preferably, it is derived from a bacterium belonging to the genus Shuwanella.

Further, “(b) a gene encoding a signal peptide consisting of the amino acid sequence described in SEQ ID NO: 1” is a 4-heme protein derived from Shewanella oneidensis MR-1 (GenBank accession number NC_004347) belonging to the genus Shewanella , as described above. Correspond to the gene encoding the signal peptide. Examples of the base sequence of this gene include those shown in SEQ ID NO: 2, but are not limited thereto, and may be a base sequence in which one to a plurality of bases are substituted in the base sequence. Can be appropriately designed and prepared according to the codon degeneracy shown in Table 1 described above. Techniques for substituting one or a plurality of bases in a known base sequence are known in the art. Examples of such methods include site-directed mutation (Site-directed Mutagenesis), restriction enzyme treatment, synthetic gene methods, The usual genetic engineering techniques such as PCR may be mentioned.

  Further, “(c) foreign protein structural gene” means a gene that is not possessed by a bacterium belonging to the genus Shuwanella used as a host in the present invention, that is, a gene encoding a protein that is not originally produced by the cell. As long as this is the case, the origin of prokaryotic organisms, eukaryotic organisms, plants, animals (including vertebrates such as humans, and invertebrates such as insects), microorganisms, etc. is not particularly questioned. In terms of industrial value, protein structural genes are preferably derived from eukaryotes, particularly higher organisms such as animals (preferably vertebrates including humans) and plants.

  More preferred foreign proteins include heme proteins that have been difficult to produce by genetic recombination methods using prokaryotic cells such as E. coli and eukaryotic cells such as yeast. Heme proteins are proteins that have iron porphyrin complexes (heme) in their molecules. These hemoproteins include, for example, hemoglobin and myoglobin involved in the transport and storage of enzyme molecules, and peroxidase, catalase, Cytochrome p-450, cytochromes involved in electron transfer, etc. are included. Cytochromes are preferred. The cytochromes include cytochrome a (formyl porphyrin iron) such as cytochrome a1 and cytochrome a3; cytochrome b (prototoporphyrin iron) such as cytochrome b2, cytochrome b5, cytochrome b559 and cytochrome b563, depending on the type of heme contained; Cytochrome c (mesoporphyrin derivative iron) such as c1, c2, c6, c551 and c553 and cytochrome f; and cytochrome d (dihydroporphyrin derivative iron). Preferred is cytochrome c derived from a eukaryote, particularly human cytochrome c derived from a human (SEQ ID NO: 3).

  Other suitable foreign proteins include proteins that are affected by proteases in cytoplasmic expression, such as protein kinases, or proteins that have disulfide bonds within or between molecules.

  The “(c) foreign protein structural gene” encoding these foreign proteins may have a base sequence originally possessed by the organism from which it is derived, but it can be any one that encodes its amino acid sequence. Alternatively, it may be designed using a codon frequently used in a bacterium belonging to the genus Shuwanella used as a host. For example, the natural gene encoding the amino acid sequence of human cytochrome c (SEQ ID NO: 3) has the base sequence shown in SEQ ID NO: 4, but is not limited thereto, and has, for example, the base sequence shown in SEQ ID NO: 5. May be. This base sequence is a base sequence that encodes the human cytochrome c amino acid sequence artificially designed based on Table 2 described above according to the codon usage of the host (bacteria belonging to the genus Shuwanella) used for genetic recombination. It is.

  The “(c) foreign protein structural gene” has a continuous amino acid frame at the 5 ′ end thereof and (b) the 3 ′ end of a gene encoding a signal peptide consisting of the amino acid sequence described in SEQ ID NO: 1. Thus, the foreign protein can be expressed and produced as a mature protein having normal structure and activity in the periplasm of bacteria belonging to the genus Shuwanella.

  The “gene cassette for expressing foreign protein” of the present invention can be constructed according to genetic engineering techniques and molecular biological experimental procedures that are generally widely used in the art. For example, J., Sambrook, E., F., Frisch, T., Maniatis, published by Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory press, 1989 And D., M., Glover, DNA cloning, IRL publication, 1985, and the like.

  Further, according to the “gene cassette for expressing foreign protein” of the present invention, by incorporating this into an existing vector (plasmid), the above-mentioned foreign protein is expressed and produced in the periplasm of bacteria belonging to the genus Schwanella. A "foreign protein expression vector" can be prepared, but the operation can also be performed according to the above-described conventional genetic engineering techniques and molecular biological experimental procedures.

A transformant can be prepared by introducing the expression vector thus prepared into a bacterium belonging to the genus Shuwanella. Specific examples of the bacterium belonging to the genus Shuwanella include those described above. Preferably, Shewanella frigidimarina, Shewanella putrefaciens, Shewanellajaponica , a Shewanella oneidensis, more preferably Shewanella oneidensis, a inter alia Shewanella oneidensis MR-1.

  The transformant thus obtained can then be cultured in a suitable medium depending on the host (bacteria belonging to the genus Shuwanella) in order to produce the desired foreign protein. Note that the transformant is also an object of the present invention, and the present invention also provides such a transformant.

The medium contains a carbon source, a nitrogen source, an inorganic substance, a vitamin, a drug and the like essential for the growth of the transformant. Examples of the medium include LB medium (Nissui Pharmaceutical), chemical synthesis medium (Takayama, Y., Shen, Y., and Akutsu, H. (2007) J. Biochem . 141, 121-126), 2xYT medium ( Molecular Cloning), CHL medium (Takayama, Y., Kobayashi, Y., Yahata, N., Saitoh, T., Hori, H., Ikegami, T., and Akutsu, H. (2006) Biochemistry 45, 3163- 3169). A 2 × YT medium is preferable.

  The culture is usually carried out in the temperature range of 4 to 30 ° C. for about several to 360 hours, and aeration and agitation can be added as necessary. The culture temperature is preferably 25 to 30 ° C.

  The production method of the present invention is further carried out by collecting the target protein from the culture thus obtained. The target foreign protein can be collected by extracting the target foreign protein accumulated in the periplasm of the transformant (bacteria belonging to the genus Shuwanella) by a known method after culture, and purifying it as necessary. It can be carried out. At this time, the host bacteria obtained by the culture may be disrupted, whereby the target foreign protein accumulated in the periplasm can be taken out into the culture supernatant. Separation and purification can be performed by, for example, salting out, solvent precipitation, dialysis, ultrafiltration, gel electrophoresis, or gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, affinity chromatography, etc. Can be combined.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, the following Example does not limit this invention at all. The genetic engineering technique and molecular biological experimentation used in the present invention are generally performed by methods widely used, for example, molecular cloning by J., Sambrook, E., F., Frisch, T., Maniatis. Second edition (Molecular Cloning 2nd edition), published by Cold Spring Harbor Laboratory press, 1989, by D., M., Glover, DNA cloning, IRL published, 1985, etc. Can be done according to the method.

Example 1 Production of human cytochrome c (1) Construction of human cytochrome c expression vector (see FIGS. 1 to 7)
A gene (GeneID: 1170426, locus tag: SO2727) encoding a 4-heme protein derived from the host Shewanella oneidensis MR-1 (GenBank accession number NC_004347) was obtained by genomic PCR (FIG. 1). In FIG. 1, a bold italic part is a coding region of a 4-heme protein derived from Shewanella oneidensis MR-1, and an underlined region located in front of it is a region encoding a signal peptide (signal sequence).

Next, in FIG. 1, recognition sites for restriction enzyme sites Pst I and Eco RI were introduced into the portions enclosed by squares, respectively, and these were converted into restriction enzyme sites (Pst I and Eco RI) of pKF19k DNA (GenBank accession number D63847). ) (FIG. 2). Incidentally, pKF19k DNA has a double-amber mutation on the kanamycin resistance gene (Km ^r am2) is a pUC-type vectors. The base sequence of pKF19k registered in the above GenBank is 2,203 bp with deletion of C before the Nde I site, but 2,204 bp with C before the Nde I site is Takara. It can be obtained as pKF19k-2 DNA from biotechnology. Host (Shewanella oneidensis MR-1) in a back to work the kanamycin resistance gene (Km ^r am2) contained in the pKF19k plasmid, based on the two amber codons in the gene (Gene 1995, 152, 271-275) A vector (pKF19STC) containing a gene of 4 heme protein derived from Shewanella oneidensis MR-1 was constructed. Next, in order to use this signal sequence (underlined part), the front and back three bases (GCCGCG), which is the boundary between the signal sequence (underlined part) and the coding region (bold italic part) of the 4 heme protein, GCC ↓ GGC and A Nae I site was introduced so as to cut at the blunt end (pKF19sig vector).

On the other hand, a cytochrome c gene (GeneID: 54205) derived from human mitochondria was subjected to PCR with a primer phosphorylated at the 5 'end in line with the codon usage and GC content of the host ( Shewanella oneidensis MR-1). . The base sequence of the gene encoding human cytochrome c thus prepared is shown in FIG. FIG. 3 compares the original base sequence of the human cytochrome c gene (top) with the above-mentioned base sequence (bottom) adjusted for codons. Both were identical at the rate of 93.02% (293/315). Both encode the amino acid sequence of human cytochrome c (protein) shown in FIG.

It processes the DNA obtained above PCR with Eco RI site at the 3 'terminus has the Eco RI site, was prepared a DNA containing the gene for human cytochrome c (Fig. 5). In FIG. 5, the square box indicates the Eco RI site.

The pKF19sig vector prepared above was digested with restriction enzymes Nae I and Eco RI and purified, and then the DNA containing the above human cytochrome c gene (FIG. 5) was ligated (FIG. 6). Thus, a human cytochrome c expression vector (pKF19signalHcyc) containing the DNA sequence shown in FIG. 7a was constructed (FIG. 7b).

(2) Production of human cytochrome c
The plasmid pKF19signalHcyc was transformed into the host Shewanella oneidensis MR-1 (American Type Culture Collection number: BAA-1096, obtained from Dr. Nealson KH, a member of the California Institute of Technology in this experiment) using electroporation. The obtained transformant was cultured at 30 ° C. overnight in an LB plate medium containing 10 μg / ml rifampicin and 100 μg / ml kanamycin. Single colonies were transferred to 3 ml of test tube medium (LB medium containing 10 μg / ml rifampicin and 100 μg / ml kanamycin) and cultured overnight at 30 ° C. with microaerobic shaking. 1 ml of the culture solution was transferred to a 100 ml flask medium (LB medium containing 10 μg / ml rifampicin and 100 μg / ml kanamycin), and cultured with shaking at 30 ° C. until the OD ₆₀₀ reached 0.5. 100 ml of the culture solution was transferred to a 1 L large flask medium (2 x YT medium containing 10 μg / ml rifampicin and 200 μg / ml kanamycin) and cultured overnight at 30 ° C with microaerobic shaking. . Cytochrome c has heme as a prosthetic group. Therefore, whether or not cytochrome c is expressed can be easily determined by visual observation based on whether or not the bacterial body becomes pink.

Cytochrome c was purified at 4 ° C. Specifically, the cells expressing cytochrome c were disrupted with ultrasonic waves, centrifuged (70,000 g, 1 hour), the supernatant was taken, and 50% ammonium sulfate fraction was obtained. The supernatant was dialyzed and desalted, and then purified by cation exchange column chromatography. The column used was a Q-Sepharose column (26/10) manufactured by Amersham Pharmacia. Elution was performed with a phosphate buffer (pH 7.4) with a sodium chloride gradient of 0 to 300 mM. Cytochrome c was eluted at a salt concentration of approximately 250 mM. For purification, SDS-polyacrylamide gel electrophoresis and visible ultraviolet absorption spectrum (see 3-2) were used to obtain a ratio of reduced 410 nm and oxidized 280 nm, and a purity of 4.66 was confirmed. 410nm indicates absorption of heme, 280nm indicates absorption including other proteins, and cytochrome c is known to be sufficiently purified at a value of 4.45 or more (Jeng, W.-Y., Chen, C.- Y., Chang, H.-C., and Chuang, W.-J. 2002, J. Bioenergetics and Biomembranes Vol. 34, pp. 423-431). As a result, 11 mg of cytochrome c was obtained per liter of the medium. The yield was calculated from the extinction coefficient e ₄₁₀ = 106.1 mM ^-1 cm ^-1 of the oxidized form 410 nm and the extinction coefficient e ₄₁₆ = 129.1 mM ^-1 cm ^{-1 of the} reduced form 416 nm.

(3) Confirmation of function of human cytochrome c The human cytochrome c obtained by the above method was subjected to the following test to confirm the function and structure.

(3-1) Amino acid sequence The cytochrome c desalted and purified as described above was subjected to amino acid sequencing according to a conventional method. As a result, the amino acid sequence of the N region 15 residues of human cytochrome c obtained in (2) above was GDVEKGKKIFIMKxS (x is unknown). The 14th amino acid was not detected here, but was identified as Cys by mass spectrometry as described later. Since the Cys could not be detected by amino acid analysis, it was considered to be covalently bound to heme. Thus, this sequence is the amino acid sequence of the N region 15 residues of the amino acid sequence of human cytochrome c (GenBank accession number: NP — 061820; Matsubara, H. et al., J. Biol. Chem, 238, 2732, 1963). It was confirmed to be the same.

(3-2) Redox characteristics (Fig. 8)
The cytochrome c obtained above was air oxidized. The air-oxidized cytochrome c solution (1.8 mM) was placed in a 1 mm long quartz cell and measured with a Beckman DU-640 spectrometer. Cytochrome c was reduced by adding a small amount of dithionite sodium salt to the cytochrome c solution.

  The results are shown in FIG. As can be seen from this result, the human cytochrome c obtained in (2) above has a characteristic of the Soray band characteristic of the heme protein, the α and γ bands in the oxidized form, and the α, β and γ bands in the reduced form. It showed the behavior of appearing. From this, it was confirmed that the human cytochrome c has redox ability.

(3-3) Mass spectrometry (Figure 9)
1 μl of the cytochrome c solution desalted and purified as described above was mixed with 1 μl of matrix (10 mg / ml sinapinic acid dissolved in 50% acetonitrile and 0.1% trifluoroacetic acid solution) and placed on a plate for mass spectrometry. This was analyzed using a MALDI-TOF mass spectrometer (Autoflex, Bruker).

  The results are shown in FIG. As can be seen from this result, the mass (actually measured value) of human cytochrome c obtained in (2) above was about 12,224. In calculation, the total mass of human cytochrome c peptides (315 aa) (11,586) and heme (620) is 12,206, which is almost the same as the actual measured value. Therefore, the human cytochrome obtained above is used. c was determined to be a heme protein with covalently bound heme. Further, from this result, it was found that the human cytochrome c obtained in (2) has no mutation such as amino acid substitution, deletion and insertion. From this, it is considered that the 14th amino acid from the N-terminus that could not be detected by the amino acid sequence of (3-1) was Cys and could not be confirmed because heme was bound here.

(3-4) NMR analysis (Figure 10)
A 0.5 mM cytochrome c solution dissolved in 30 mM phosphate buffer was lyophilized and the solvent was replaced with heavy water. At this time, a sample that spontaneously oxidizes was placed in a 5 mm NMR tube and measured using a Bruker Avance DRX-600 spectrometer. The sample was reduced by adding a small amount of dithionite sodium salt to the sample.

  The results are shown in FIG. Signals have already been assigned to NMR of human cytochrome c (BMRB5406). The results shown in FIG. 10 were consistent with this. In particular, the coordinated methionine signal (reduced form) and the heme methyl signal (oxidized form) are signals characteristic of human cytochrome c, but the human cytochrome c obtained in (2) above. Had this signal as well. From this, it was confirmed that the human cytochrome c obtained in the above (2) similarly holds the structure of human-derived natural cytochrome c.

  From the results of (3-1) to (3-4) above, the recombinant human cytochrome c produced by the method of the present invention has the same structure and function as natural cytochrome c obtained from humans. It can be determined that it is a protein.

Example 2 Production of Calcium Dependent Protein Kinase 2 (CDPK2) Construction of CDPK2 Expression Vector (See FIGS. 11-12)
Primer (5'-pGCCGAACCAAAACCAGCAACTGAGCCCAAG-3 [primer with 5 'end phosphorylated] (SEQ ID NO: 11), 5'-TTGGT GAATTC TTATAAGATTTCTTCACTC-3' (SEQ ID NO: 12) [underlined region is Eco RI site]) Then, DNA containing a gene encoding calcium-dependent protein kinase 2 (CDPK2) derived from Solanum tuberosum (potato) was obtained by PCR. The base sequence of the gene encoding potato-derived CDPK2 (cdpk2) is registered in GenBank AB051809 (the 12-15025 region is a structural gene) (FIG. 11) (SEQ ID NOs: 9 and 10). The DNA obtained by the PCR was treated with Eco RI to prepare a DNA containing a gene (cdpk2) encoding CDPK2 derived from potato having an Eco RI site at the 3 ′ end ([2] in FIG. 12). ).

The pKF19sig vector prepared in Example 1 (1) was purified by digestion with restriction enzymes Nae I and Eco RI, and then ligated with DNA containing the gene encoding CDPK2 (cdpk2) (FIG. 12). Thus, a CDPK2 expression vector (pKFcdpk2sig) in which cdpk2 was inserted downstream of the signal sequence was constructed (FIG. 13).

(2) Production of CDPK2 As in Example 1 (2), the plasmid pKFcdpk2sig [pKF19signalHcyc was used in Example 1 (2)] was transformed into the host Shewanella oneidensis TSP-C by electroporation. The transformant was cultured overnight at 30 ° C. in LB plate medium containing 10 μg / ml rifampicin and 100 μg / ml kanamycin. Single colonies were transferred to 3 ml of test tube medium (LB medium containing 10 μg / ml rifampicin and 100 μg / ml kanamycin) and cultured overnight at 30 ° C. with microaerobic shaking. 1 ml of the culture solution was transferred to a 100 ml flask medium (LB medium containing 10 μg / ml rifampicin and 100 μg / ml kanamycin) and cultured overnight at 30 ° C. with microaerobic shaking. This was sonicated, centrifuged, and separated into a supernatant and a precipitate, and protein expression was confirmed by SDS-polyacrylamide electrophoresis.

(3) Confirmation of CDPK2 expression The collected cells of (2) were centrifuged after sonication, separated into supernatant and precipitate, and subjected to SDS-polyacrylamide electrophoresis at 10% acrylamide concentration. The gel was stained with Coomassie Brilliant Blue dye.

The results are shown in FIG. The expression level of CDPK2 was compared by Western blotting between the microbial cells (+ vec) into which the CDPK2 expression vector (pKFcdpk2sig) was introduced and the non-introduced microbial cells (-vec). In the figure, “+ vec1” and “+ vec2” indicate the results of experiments on different colonies. “+ Vec1 & Mag” indicates the result of culturing the transformant with 0.5 mM MgCl ₂ added thereto. The cells cultured after introducing each vector were disrupted and separated into a supernatant (soluble fraction: sup) and a precipitate (insoluble fraction: pellet) and subjected to SDS-PAGE, as shown in FIG. In addition, the expression of CDPK2 was confirmed in the soluble fraction (detected molecular weight: 59 kDa).

(3) Confirmation of CDPK2 functions
CDPK2 is autophosphorylated by ATP and Ca ²⁺ . Therefore, the function of the CDPK2 produced above was evaluated by measuring the autophosphate ability. Specifically, ProQ diamond gel stain method (Takeda, H. et al, Rapid Communications in Mass Spectrometry 17 (8), 2075 -081 (2003); Kinoshita, M. et al. Dalton Transactions 1189-1193 (2004) Kinoshita E. et al. Journal of Separation Science 28 (2) 155-162 (2005)) was used to measure autophosphorylation. The measurement of autophosphorylation was commissioned to PerkinElmer Japan.

  The results are shown in FIG. From this result, it was confirmed that CDPK2 obtained above has autophosphorylation ability. Further, from this molecular weight, it was confirmed that the signal peptide used in the production was cleaved, and mature potato-derived CDPK2 was obtained.

This shows the base sequence of the gene encoding 4 heme protein derived from Shewanella oneidensis MR-1. In the figure, the bold italic part is the coding region of the 4-heme protein derived from Shewanella oneidensis MR-1, and the underlined region located in the downstream region is a region encoding a signal peptide (signal sequence). In the boxed area, the 5 ′ end is the Pst I recognition site and the 3 ′ end is the Eco RI recognition site. It is a conceptual diagram which shows construction | assembly of the vector (pKF19STC) containing the gene which codes 4 heme protein derived from Shewanella oneidensis MR-1. It is the figure which contrasted the natural origin base sequence (upper part) of the gene which codes human type | mold cytochrome c, and the base sequence created in Example 1 (1) (lower part). The amino acid sequence of human-type cytochrome c is shown. The base sequence (dashed line part) of the gene which codes the human type | mold cytochrome c created in Example 1 (1) is shown. The part enclosed by a square is a recognition site for Eco RI added to the 3 'end. It is the schematic which shows the ligation method of DNA containing a human type | mold cytochrome c gene to a pKF19sig vector. (1) Digest the insertion part of pKF19STC with Nae I and Eco RI [1]. (2) Phosphorylate the 5 'end of human cytochrome c gene and digest with Eco RI [2]. Next, (3) The above [1] and [2] are replaced with blunt ends and Eco RI sites. The constructed human cytochrome c expression vector (pKF19signalHcyc) (Fig. B) and the DNA sequence (Fig. A) containing the human cytochrome c gene introduced therein are shown. In the sequence, the linear underlined region is a base sequence encoding a signal peptide, and the wavy line is a base sequence encoding human cytochrome c. It is a visible absorption spectrum which shows the redox characteristic of human-type cytochrome c. The mass spectrometry result of human type | mold cytochrome c is shown. The NMR analysis result of human-type cytochrome c is shown. 2 shows the amino acid sequence of calcium-dependent protein kinase 2 (CDPK2) derived from Solanum tuberosum (potato) and the base sequence of the gene (cdpk2) encoding it. It is the schematic which shows the method of ligating DNA containing the gene which codes the calcium-dependent protein kinase 2 (CDPK2) derived from Solanum tuberosum (potato) to pKF19sig vector. (1) Digest the insertion part of pKF19STC with Nae I and Eco RI [1]. (2) Phosphorylate the 5 'end of CDPK2 gene and digest with Eco RI [2]. Next, (3) The above [1] and [2] are replaced with blunt ends and Eco RI sites. The constructed CDPK2 expression vector (pKFcdpk2sigd) is shown. The result of SDS-polyacrylamide electrophoresis which confirmed the expression of CDPK2 is shown. The result of having confirmed the function (autophosphorylation ability) of CDPK2 is shown.

SEQ ID NO: 11 shows the base sequence of the sense primer used to obtain DNA containing the gene encoding CDPK2.
SEQ ID NO: 12 shows the base sequence of the antisense primer used to obtain DNA containing the gene encoding CDPK2.

Claims (11)

  Culturing a bacterium belonging to the genus Shuwanella transformed with a vector containing a gene encoding a signal peptide consisting of the amino acid sequence described in SEQ ID NO: 1 and a gene encoding a protein derived from a eukaryote, and the resulting culture A method for producing a eukaryotic protein, comprising a step of collecting a eukaryotic protein produced in the periplasm of the bacterium from a product.   2. The production method according to claim 1, wherein the gene encoding the signal peptide has the base sequence shown in SEQ ID NO: 2 or has a base sequence in which 1 to several bases are substituted in the base sequence. .   The production method according to claim 1 or 2, wherein the eukaryotic protein is a heme protein or a protein having an intramolecular or intermolecular disulfide bond.   The production method according to claim 3, wherein the heme protein is human cytochrome c. A gene cassette for expressing a foreign protein used for producing a foreign protein in the periplasm of a bacterium belonging to the genus Shuwanella, which has any one of the following base sequences (1) to (3):
(1) (a) a promoter that is expressed in a bacterium belonging to the genus Shuwanella, and a base sequence comprising a gene encoding a signal peptide consisting of the amino acid sequence described in SEQ ID NO: 1 linked downstream thereof, (b)
(2) (b) The 3 'end of the gene encoding the signal peptide consisting of the amino acid sequence described in SEQ ID NO: 1 and (c) the 5' end of the foreign protein structural gene so that the amino acid frame is continuous. A base sequence formed by ligation,
(3) (a) a promoter expressed in a bacterium belonging to the genus Shuwanella, and (b) linked to the downstream thereof (b) at the 3 ′ end of a gene encoding a signal peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, c) A nucleotide sequence in which the 5 'end of a foreign protein structural gene is linked so that the amino acid frame is continuous.   The foreign gene according to claim 5, wherein the gene encoding the signal peptide has the base sequence shown in SEQ ID NO: 2 or has a base sequence in which 1 to several bases are substituted in the base sequence. Gene cassette for protein expression.   The gene cassette for expressing a foreign protein according to claim 5 or 6, wherein the foreign protein is a heme protein or a protein having a disulfide bond within or between molecules.   The gene cassette for foreign protein expression according to claim 7, wherein the heme protein is human cytochrome c.   A foreign protein expression vector for a bacterium belonging to the genus Shuwanella, comprising the gene cassette for expressing a foreign protein according to any one of claims 5 to 8.   A bacterium belonging to the genus Shuwanella transformed with the foreign protein expression vector according to claim 9.   10. Transforming a bacterium belonging to the genus Shuwanella using the foreign protein expression vector according to claim 9, then culturing the transformed bacterium, and the foreign product produced in the periplasm of the bacterium from the obtained culture A method for producing a foreign protein, comprising a step of collecting a protein.

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