JP2008193953A - Development and application of new protein expression system using actinomyces as host - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To construct an expression system expressing an exogenous protein derived from organisms except actinomyces in the actinomyces. <P>SOLUTION: A vector expressing the exogenous protein derived from the organisms except the actinomyces in the actinomyces has a promoter sequence of a phospholipase D gene derived from Streptoverticillium cinnamoneum, a signal sequence of the phospholipase D gene derived from the Streptoverticillium cinnamoneum and linked to the downstream of the promoter so as to be expressible, an exogenous base sequence encoding the exogenous protein and linked to the downstream of the signal sequence so as to be expressible, and a terminator sequence of the phospholipase D gene derived from the Streptoverticillium cinnamoneum. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to the development and application of a new protein expression system using actinomycetes as a host.

  Actinomycetes are Gram-positive soil bacteria that always exist in aerobic soils at high density. Actinomycetes are a group of bacteria (Bacteria), but normal bacteria show only simple forms such as spheres and rods, whereas typical actinomycetes germinate when the environmental conditions for growth are in place. The hyphae are stretched and branched, then the hyphae are elongated, and the aerial hyphae eventually take a complex form of breaking into spores. Therefore, it can be said that it is closer to a filamentous fungus that is a eukaryote rather than a bacterium. Actinomycetes are also representative of antibiotic-producing bacteria, as can be seen from the fact that the majority of antibiotics on the market as pharmaceuticals are produced by actinomycetes, and a wide variety of antibiotics (secondary metabolites) Has production capacity. As a result of genome analysis, it is clear that the genome size and the amount of information (number of genes) encoded on the genome are by far the largest among bacteria, and actinomycetes are the most evolved among bacteria. It is considered a living creature.

  Although molecular biological research in actinomycetes, especially Streptomyces, has been in full swing since the 1980s, research has been conducted before that. In the mid 1950s, it was found that genetic recombination occurred in actinomycetes, and since then genetic analysis has been steadily performed by plasmid-mediated conjugation and protoplast fusion. The group studied was Hopwood et al., UK, and the strain of interest was Streptomyces coelicolor. Thereafter, this strain has become a standard bacterium for genetic and molecular biological analysis of Streptomyces (see Non-Patent Documents 1 and 4).

  With the development of recombinant DNA technology, the molecular biology of microorganisms centered on E. coli has become popular, but actinomycetes are genetically different from E. coli, and the genes of actinomycetes are expressed in E. coli. There are many things that do not. For this reason, a unique cloning system was required. Subsequently, the development of a cloning system (host-vector system) in Streptomyces by Hopwood et al. The vector is an improved plasmid of actinomycetes, and a thiostrepton resistance gene is suitable as a selection marker. As a host, S. cerevisiae. Streptomyces lividans, closely related to coelicolor, is used as standard. S. coelicolor and S.E. Although lividans is closely related to the same species, the latter is more suitable as a host for genetic recombination because it has few restrictions and has high transformation efficiency. Genes involved in enzyme production, primary metabolism, antibiotic biosynthesis, morphogenesis (differentiation), etc. have been cloned using the developed host-vector system, and molecular biological research has been actively conducted. . (See Non-Patent Documents 1, 3, and 4)

  In the laboratory of the present inventors, the gene of the secretory protein Phospholipase D (PLD) derived from Streptovertillium cinnamoneum and its upstream promoter region and downstream terminator region have been introduced into pUC702, which is an actinomycete-E. Coli shuttle vector. It has been confirmed that pUC702-promoter-PLD is constructed and recombination of Streptomyces lividans enables PLD secretion production.

  More specifically, by incorporating this promoter region, the PLD production amount was about 20 times that of the wild strain, and the specific activity was also almost the same as the PLD derived from the wild strain Streptovertillium cinnamoneum (see Non-Patent Document 2). .

  With regard to these research results, Professor Hideki Fukuda of Kobe University has applied through Hyogo TLO (see Patent Document 1). From these, it has been clarified that the PLD expression promoter derived from Streptovertillium cinnamoneum is effective and powerful in PLD production even in actinomycetes recombinants.

Japanese Patent Laid-Open No. 2002-51780 Tsutomu Hattori, New and Soil Microorganisms (9) Functions and Functions of Actinomycetes Hirotomo (2003) C. Ogino, H .; Fukuda; Over-expression system for secretory phospholipase D by Streptomyces lividans [Appl Microbiol Biotechnol 64, 823-828.2004]. C. Ogino, H .; Fukuda; Phospholipase D from Streptoverticillium cinnamonum: protein engineering and application for phospholipids production 107 [Junnal folfoids production] 2003] Tadahiko Ando, Genetic Engineering for Material Production Asakura Shoten (1985)

  As described above, although microorganisms other than actinomycetes such as E. coli have been proposed for host vector systems that express or secrete production of various heterologous genetically modified proteins, There are few reports on the expression system or secretory production system of foreign proteins derived from actinomycetes. Therefore, the expression or secretion of various useful foreign proteins, including heterologous proteins derived from mammals, has been expected in actinomycetes, but no successful examples have been reported yet.

  In addition, in the laboratory to which the present inventors belong, Phospholipase D (PLD) having a high transphosphorylation reaction has been identified from the actinomycete Streptoverticium cinnamoneum (IFO 12852) and its gene sequence has been determined, and Streptomyces lividans Although a mass secretion expression system of PLD using 1326 strain as a host was constructed (see Non-Patent Document 3), a secretory expression system capable of secreting foreign proteins derived from organisms other than actinomycetes by actinomycetes has not been constructed yet. Absent.

  The present invention has been made in view of the above circumstances, and an object thereof is to construct an expression system capable of expressing foreign proteins derived from organisms other than actinomycetes by actinomycetes.

  Actinomycetes produce various secondary metabolites and have many protein genes, so it is expected that various proteins can be expressed and expected to be used as a host for substance production. Has been. In the actinomycetes expression system of the laboratory to which the present inventors belong, mass secretion expression of PLD is already possible due to the high transcription ability of the Promoter region located upstream of the cloned PLD gene.

  Accordingly, the present inventors have established an industrially useful protein secretory production system based on the idea that various proteins can be expressed by replacing the PLD gene with genes of other proteins. First, an expression system for foreign proteins derived from organisms other than actinomycetes was constructed as a model protein, and it was investigated whether this actinomycete expression system could produce foreign proteins.

  As a result, the present inventors made trial and error to develop a general-purpose vector in order to apply the above-described expression system to the expression of various foreign-derived proteins, and finally obtained a general-purpose vector having a specific configuration. As a result, it was found that a foreign protein derived from an organism other than actinomycetes can be secreted and expressed in an actinomycetes expression system, and the present invention was completed.

  That is, according to the present invention, a vector for expressing a foreign protein derived from an organism other than actinomycetes in actinomycetes, comprising a promoter sequence of a phospholipase D gene derived from Streptobacillus cinnamonium, and a downstream of the promoter A signal sequence of a phospholipase D gene derived from Streptobacillus cinnamonium that is operably linked to the gene, and a foreign base sequence encoding a foreign protein that is operably linked downstream of the signal sequence; And a terminator sequence of a phospholipase D gene derived from Streptobacillus cinnamonium linked downstream of the foreign protein.

  According to this vector, as a phospholipase D gene (PLD gene) derived from Strep. Cinnamoneum, which is a kind of actinomycetes, the promoter region and the PLD gene are secreted into the medium. Since a foreign gene is inserted in a sequence that can be expressed as a protein downstream of the signal region using a related signal region and a terminator region present downstream of the PLD gene (ORF), secretion from the PLD gene Using the signal, the expressed foreign protein can be secreted and produced in a medium outside the actinomycetes.

  Further, according to the present invention, a vector for incorporating a foreign protein derived from an organism other than actinomycetes and expressing the foreign protein in actinomycetes, comprising a phospholipase D gene derived from Streptobacillus cinnamonium A promoter sequence, a signal sequence of a phospholipase D gene derived from Streptobacillus cinnamonium that is operably linked downstream of the promoter, and a foreign protein encoding a foreign protein provided downstream of the signal sequence There is provided a vector comprising a cloning site for inserting a base sequence and a terminator sequence of a phospholipase D gene derived from Streptobacillus cinnamonium, which is linked downstream of the cloning site.

  According to this vector, as a phospholipase D gene (PLD gene) derived from Strep. Cinnamoneum, which is a kind of actinomycetes, the promoter region and the PLD gene are secreted into the medium. Using a related signal region and a terminator region present downstream of the PLD gene (ORF), a cloning site for inserting a foreign gene in a sequence that can be expressed as a protein is provided downstream of the signal region. Therefore, by inserting a foreign base sequence encoding a foreign protein derived from an organism other than actinomycetes into the cloning site in a form that can be expressed, the expressed foreign protein can be introduced into the cloning site using a secretory signal derived from the PLD gene. Secretory production in the medium outside the cell Possible to become.

  Further, according to the present invention, a recombinant actinomycete for expressing a foreign protein derived from an organism other than actinomycetes, wherein the above-described vector having a foreign base sequence already inserted therein is incorporated. Is provided.

  According to this recombinant actinomycetes, a promoter region and a PLD gene into a medium as a phospholipase D gene (PLD gene) derived from Strep. Cinnamoneum, which is a kind of actinomycetes. Using a signal region related to secretory expression and a terminator region located downstream of the PLD gene (ORF), a vector into which a foreign gene is inserted in a sequence that can be expressed as a protein is incorporated downstream of the signal region. Therefore, the expressed foreign protein can be secreted and produced in a medium outside the cells of actinomycetes using a secretion signal derived from the PLD gene.

  According to the present invention, there is also provided a method for producing a foreign protein derived from an organism other than actinomycetes in actinomycetes, the step of culturing the above recombinant actinomycetes in a medium, and the cells of the recombinant actinomycetes Or obtaining a foreign protein from the culture medium.

  According to this method, as a phospholipase D gene (PLD gene) derived from Stv. Cinnamoneum which is a kind of actinomycetes, the promoter region and the PLD gene are secreted into the medium. A radiation in which a vector in which a foreign gene is inserted in a sequence that can be expressed as a protein is incorporated downstream of the signal region using a related signal region and a terminator region present downstream of the PLD gene (ORF). Since the bacterium is used, the expressed foreign protein can be secreted and produced in a medium outside the cells of actinomycetes.

  According to the present invention, foreign proteins derived from organisms other than actinomycetes can be secreted and produced by actinomycetes in a medium outside the actinomycetes cells.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all the drawings, the same reference numerals are given to the same components, and the description will be omitted as appropriate.

<Overview>
The vector according to the present embodiment is a vector that expresses a foreign protein derived from an organism other than actinomycetes in actinomycetes, and comprises a promoter sequence of a phospholipase D gene derived from Streptobacillus cinnamonium, and downstream of the promoter A signal sequence of a phospholipase D gene derived from Streptobacillus cinnamonium that is operably linked to the gene, and a foreign base sequence encoding a foreign protein that is operably linked downstream of the signal sequence; And a terminator sequence of a phospholipase D gene derived from Streptobacillus cinnamonium, which is linked downstream of the foreign protein.

  According to this vector, as a phospholipase D gene (PLD gene) derived from Strep. Cinnamoneum, which is a kind of actinomycetes, the promoter region and the PLD gene are secreted into the medium. Since a foreign gene is inserted in a sequence that can be expressed as a protein downstream of the signal region using a related signal region and a terminator region present downstream of the PLD gene (ORF), secretion from the PLD gene Using the signal, the expressed foreign protein can be secreted and produced in a medium outside the actinomycetes.

<Promoter>
The promoter used in the present embodiment is a phospholipase D (hereinafter referred to as Phospholipase D), hereinafter referred to as Streptovertylium cinnamonium (IFO12852) (Streptovertillium cinnamonum, hereinafter abbreviated as Stv. Cinnamonum (IFO12852)). It is a gene promoter.

  Among the actinomycetes group, it has been found that Streptoverticillium cinnamonium (IFO12852) (Streptovertillium cinnamonum, hereinafter abbreviated as Stv. Cinnamonum (IFO12852)) is the most highly secreted PLD activity strain at present. (Nakajima et al., Biotechnol. Bioeng., Vol. 44, 1193-1198 (1994)). In addition, about Streptomycesium, it may be classified into Streptomyces (Streptomyces) on classification.

The nucleotide sequence of a clone containing the promoter sequence, structural gene sequence, and terminator sequence of the phospholipase D gene derived from Streptobacillus cinnamonium is shown in SEQ ID NO: 4. The base sequence from base number 38 to base number 1407 in the sequence listing of SEQ ID NO: 4 is the promoter sequence (SEQ ID NO: 1) of the phospholipase D gene derived from Streptoverticillium cinnamonium.

  In the promoter used in this embodiment, one or several base sequences are deleted or substituted in the base sequence from base number 38 to base number 1407 (SEQ ID NO: 1) in the sequence listing of SEQ ID NO: 4. Alternatively, it may be an added base sequence. In this case as well, it is considered that the expression efficiency in actinomycetes is similarly excellent from the high homology of both sequences.

  Further, the promoter used in the present embodiment has a homology of 80% or more, 90% or more with the base sequence (SEQ ID NO: 1) from base number 38 to base number 1407 in the sequence listing of SEQ ID NO: 4, or The base sequence may be 95% or more. In this case as well, it is considered that the expression efficiency in actinomycetes is similarly excellent from the high homology of both sequences. In addition, about the homology defined in this specification, it shall follow the numerical value of the homology calculated by NCBI BLAST (blastn) (http://www.ncbi.nlm.nih.gov/BLAST/).

<Exogenous base sequence>
The foreign base sequence used in the present embodiment may be a base sequence encoding a foreign protein derived from an organism other than actinomycetes, and any base sequence can be used. For example, a foreign base derived from a mammal or Bacillus subtilis A base sequence encoding a protein can be preferably used. Examples of the foreign protein derived from a mammal or Bacillus subtilis include, as demonstrated in Example 2 described later, one or more foreign proteins selected from the group consisting of GFP and globin; A2, B1, and B2. It can be suitably expressed. Naturally, mammals include humans.

  However, the foreign base sequences are not limited to these base sequences. For example, globin; A2, B1, and the like for complex three-dimensional structural proteins having a heme binding region such as NO synthase, chloroperoxidase, and CooA are also included. Similarly to B2, it is considered that the vector of this embodiment can be suitably expressed in actinomycetes.

<Terminator>
The vector according to this embodiment comprises a terminator sequence derived from the genome of Streptobacillus cinnamonium linked downstream of the foreign base sequence. Thereby, the productivity of foreign proteins derived from organisms other than actinomycetes can be improved.

  The terminator sequence used in the present embodiment uses a terminator sequence derived from the genome of Streptobacillus cinnamonium because of its high expression efficiency. In the sequence listing of SEQ ID NO: 4, the base sequence from base number 3031 to base number 3213 (SEQ ID NO: 2) is the terminator sequence of the phospholipase D gene derived from Streptobacillus cinnamonium.

  Further, the terminator used in the present embodiment has a deletion of one or several base sequences in the base sequence from base number 3031 to base number 3213 (SEQ ID NO: 2) in the sequence listing of SEQ ID NO: 4. It may be a substituted or added base sequence. In this case as well, it is considered that the expression efficiency in actinomycetes is similarly excellent from the high homology of both sequences.

  Further, the terminator used in the present embodiment has a homology of 80% or more, 90% or more with the base sequence (SEQ ID NO: 2) from base number 3031 to base number 3213 in the sequence listing of SEQ ID NO: 4. Alternatively, the base sequence may be 95% or more. In this case as well, it is considered that the expression efficiency in actinomycetes is similarly excellent from the high homology of both sequences.

<Signal sequence>
The vector of this embodiment is a phosphine derived from Streptobacillus cinnamonium as a signal sequence encoding a signal peptide for secreting a foreign protein outside the cell of actinomycetes downstream of the promoter and upstream of the foreign base sequence. The signal sequence of the flipase D gene is provided. In this case, the presence of the signal peptide makes it possible to secrete the foreign protein outside the actinomycetes cell. As a result, the production efficiency of the foreign protein is improved, and it becomes easy to purify the foreign protein secreted outside the cells.

  In addition, the signal sequence of the phospholipase D gene of Streptobacillus cinnamonium is the nucleotide sequence (SEQ ID NO: 3) from nucleotide number 1408 to nucleotide number 1509 in the sequence listing of SEQ ID NO: 4. However, the signal sequence is not limited to this sequence, and the signal sequence of the phospholipase D gene derived from Streptoverticium cinnamonium as long as it can be secreted outside the cell in actinomycetes. Even if it is partially modified, it can be used.

<Replication origin array>
The vector of this embodiment may further comprise an origin of replication sequence that allows the vector to replicate in actinomycetes. In this case, the vector can be replicated in the cells of actinomycetes, and the vector can be prevented from dropping due to the growth of actinomycetes. Moreover, since the number of copies of the vector in the cells of actinomycetes can be increased, the ability to produce foreign proteins derived from organisms other than actinomycetes can be improved. The rep gene in FIGS. 1 and 5 corresponds to the origin of replication sequence that allows the vector to replicate in this actinomycete.

  The vector of this embodiment may further comprise an origin of replication sequence that allows the vector to replicate in E. coli. In this case, it becomes possible to carry out gene recombination etc. of the vector in E. coli and store it in E. coli, and it becomes easy to modify, modify and store the vector. The ori gene in FIGS. 1 and 5 corresponds to the origin of replication sequence that allows the vector to replicate in this E. coli.

<Marker>
The vector of this embodiment may further comprise an antibiotic resistance gene. In this case, by culturing actinomycetes in a medium containing the corresponding antibiotic, it is possible to prevent the vector from dropping from the cells of actinomycetes. Further, when actinomycetes are transformed with this vector, whether or not they are actually transformed can be confirmed by culturing actinomycetes in a medium containing a corresponding antibiotic. The Amp ^r gene in FIGS. 1 and 5 corresponds to this antibiotic resistance gene. However, in addition to the Amp ^r gene, any antibiotic resistance gene generally used in actinomycetes can be used. In addition, marker genes other than antibiotic resistance genes may be used.

<Cloning site>
The vector according to this embodiment has a cloning site for inserting a foreign base sequence encoding a foreign protein provided downstream of the promoter in a state before inserting the foreign base sequence encoding the foreign protein. ing. Since this cloning site is a site that is uniquely cleaved by a specific type of restriction enzyme (or combination thereof), the target foreign site is cleaved by the specific type of restriction enzyme (or combination thereof). A foreign base sequence encoding a protein can be inserted.

  In FIG. 5, the vector in the state before inserting the foreign base sequence encoding this foreign protein corresponds to pUC18 + promoter + term, and the cloning sites are sites containing NheI 1871, BamHI 1877 and BglII 1883. However, the cloning site is not limited to this sequence, and a combination of restriction enzyme sites that does not cleave sequences other than the cloning site of the vector in the state before inserting the foreign base sequence encoding this foreign protein such as pUC18 + promoter + term. Any sequence that contains can be preferably used.

  In addition, the configuration of the vector in the state before inserting the foreign base sequence encoding the foreign protein is not limited to the configuration of pUC18 + promoter + term constructed based on pUC18, and can be used in other actinomycetes. Even those prepared by modifying the vector in the same manner can be suitably used. Although it is a snake foot, the vector described in JP-A-2002-51780 does not have an appropriate cloning site as it is, and therefore it is difficult to insert a foreign base sequence encoding a foreign protein.

<Recombinant actinomycetes>
The recombinant actinomycetes of this embodiment are recombinant actinomycetes for expressing foreign proteins derived from organisms other than actinomycetes, and are recombinant actinomycetes in which the above-described vectors are incorporated. As the actinomycetes, any kind of actinomycetes may be used, for example, Streptomyces lividans (Streptomycins lividans 1326 strain, hereinafter also abbreviated as S. lividans) excellent in protein production ability is preferable. Can be used for

  It is possible to produce foreign proteins derived from organisms other than actinomycetes in actinomycetes by culturing the recombinant actinomycetes in this manner in a medium and obtaining the foreign protein from the cells or medium of the recombinant actinomycetes it can. The culture method at this time is not particularly limited, and any common actinomycete culture method can be used, and any common actinomycetes medium can be used. For example, aerobic shaking culture may be performed using LB medium. And if a foreign protein is acquired from a microbial cell or a culture medium, and it refine | purifies with the general protein purification method, foreign protein derived from organisms other than active actinomycetes with high purity can be produced.

<About Streptomyces vector>
Actinomycete plasmids used in vectors Many plasmids have been isolated from actinomycetes, and among them, selection vectors are assigned and cloning vectors capable of expressing information are determined. SCP2, SLP1, and pIJ101 These are only derived from the three types of plasmids (see Non-Patent Document 3). Table 1 shows the origin, function, molecular weight, copy number, host range of these plasmids and representative vectors constructed. Both of these are Hopwood, D .; A. Group and Cohen, S .; N. Developed by the group. Among them, the function map of pIJ101 was examined in detail by deletion mutation and foreign DNA insertion experiments, and plasmid conjugation transfer, pock formation (spread), and copy number stability (stability) were determined. (FIG. 9).

There are currently 9 types of selectable markers derived from actinomycetes incorporated into the selectable marker actinomycete plasmid used in vectors (see Non-Patent Document 3).
Selection marker
Strain-derived selection marker coelicolor A3 (2) Methylenomycin resistance (mmy)
S. fradiae neomycin resistance (aph)
S. fradiae neomycin resistance (eaac)
S. azureus thiostrepton resistance (tsr)
S. Vinaces Biomycin resistance (vph)
S. erythreus MLS resistance (mls)
S. lividans B-galactosidase (B-gal)
S. antibiotics Melanin production (mel)
S. grieseus p-aminobenzoic acid synthase (pab)

Vector pIJ702 constructed in previous studies
A number of vectors derived from the above-mentioned pIJ101 have been produced, and among them, pIJ702 has two types of selection markers (tsr, mel). The host range of pIJ702 spans many species of the genus Streptomyces, the copy number is 40 to 300, and the conjugation transmission ability is lost. The expression of the mel gene is inducible (see Non-Patent Document 3).

pUC702
An actinomycete and E. coli shuttle vector pUC702 synthesized from the actinomycetes vector pIJ702 and the E. coli vector pUC19 by the restriction enzymes SecI and KpnI has been constructed (Tobias Kieser, Mervin J. Bibb, Mark J. Buttner, Keith F.Chater). A. Howood; See Practical Streptomyces Genetics [The John Innes Foundation, Pricing by Crows, Norwich, England. 2000] (FIG. 1).

pUC702-pro-PLD
Stv. By introducing the PLD gene obtained from the genome of cinnamoneum and the surrounding promoter region, signal region, and terminator region into pUC702, a plasmid for mass secretion expression of PLD has been constructed (see non-patent document 2) (see FIG. 2). 1).

Stv. Characteristics of cinnamonium PLD-derived promoter Transcription of actinomycetes genes hardly occurs in E. coli and the like due to the specificity of the promoter sequence due to its high GC content. Although the structures of various actinomycetes promoters have been clarified, there are only a few examples of correspondence with the σ factor which is a protein expression system factor of E. coli (Non-patent Document 1 and Tsutomu Hattori, Shin Soil microorganisms (3) genes and soil microorganisms (see Hirotosha (1998)). However, a region TTTAAGGATG corresponding to Ribosome Binding Site (rbs) has been confirmed.

  In the laboratory to which the present inventors belong, a pUC702-promoter-PLD in which a PLD gene derived from Streptovertillium cinnamoneum and a promoter gene region upstream thereof is introduced into a plasmid pUC702 plasmid of E. coli and actinomycetes has been constructed so far. It has been incorporated into (Streptomyces lividans) and analyzed for its expression (FIG. 2). As a result, it has been clarified that the action of the promoter region is greatly involved in the large-scale protein expression by actinomycetes. Note that the base sequence shown in FIG. 2 corresponds to base numbers 1410 to 1620 of the base sequence of SEQ ID NO: 4. The amino acid sequence shown in FIG. 2 is represented by SEQ ID NO: 5.

All proteins released outside the cell, including proteins that form the structure outside the cell for protein secretion, are called secreted proteins because they pass through the cell membrane. During the evolution of organisms, the secretory mechanism of proteins has differentiated and has been highly developed. Therefore, the mechanism by which secretory proteins pass through the cell membrane of prokaryotic cells is similar to the mechanism by which they pass through the endoplasmic reticulum membrane in eukaryotes. Secretion of macromolecules such as proteins is blocked as much as possible by the cell membrane, as in the case of taking in other macromolecules. Basics of protein engineering (see Tokyo Kagaku Doujin (2004)).

  When secreted proteins are synthesized on ribosomes in cells, they are synthesized with an extra amino acid sequence added to the N-terminal side. The sequence is called a signal peptide, and is composed of three parts, an N region, an H region, and a C region (the PLD signal region in FIG. 2). The N region is positively charged and is associated with fusion with the membrane. The H region located in the center forms an α-helix structure, deeply penetrates the cell membrane and plays a role in smoothly passing the subsequent amino acid sequence that forms the original protein. The last C region has a region called Ala-X-Ala and is said to be recognized and cleaved by signal peptidase (SPase) (see Hiroshi Matsuzawa, Foundation of Protein Engineering, Tokyo Chemical Doujin (2004)) .

  There are various reports on the secretory mechanism of proteins, and it is reported that there are at least four secretory mechanisms in prokaryotes such as actinomycetes. Among them, the main mechanisms are Secret-dependent pathway (Sec-pathway), Signal Recognition Particle pathway (SRP-pathway), and Twin Arginine Transfusion system mechanism type T It is said.

  Many secreted proteins are said to be secreted by Sec-pathway (FIG. 3). This secretory mechanism is adapted for proteins with a signal of 18-26 amino acids. In this secretory mechanism, only unfolded proteins can cross the membrane, so that they are folded after passage. In this secretory mechanism, first, a protein in which the intracellular chaperone SecBp is translated is wound and kept in an unfolded state. The protein is transported to the membrane by binding SecBp wrapped with the protein to SecAp that is bound to the membrane protein SecYEGp. The membrane protein SecYEGp is then passed through and folded. Thereafter, the signal and protein are separated by the action of SPase located outside the cell membrane (Fernando A. Agroveres, J. Fred Dice; Protein translocation analysis membranes [see Biochimica et Biophysica Acta 1513, 1-24. 200). .

  Although there are few reports, a secretory mechanism called SRP-pathway (FIG. 3) exists. This is the case for signals with strong hydrophobicity. In this secretory mechanism as well, in order to pass the membrane protein SecYEGp, the protein passes in an unfolded state. In this secretory mechanism, SRP binds as soon as the signal region is translated. When SRP binds to FtsY on the cell membrane, ribosomes and mRNA are transported on the cell membrane. Since the protein is translated in the vicinity of SecYEGp, protein synthesis and secretion are performed simultaneously. Signals and proteins are separated by the action of SPase after the synthesis and secretion are completed (Fernando A. Agroveres, J. Fred Dice; Protein translocation analysis membranes [see Biochimica et Biophysica Acta 1513, 1-24. 200). .

  A secretory mechanism that passes through a membrane protein called TatA-D without using SecYEGp is called a TAT system. This applies to signals of 48 amino acids or more. The signal has the sequence (SRRXFLK (SEQ ID NO: 6)) in common, and it is said that twin arginine motif prevents the function of Sec-pathway. In this secretory mechanism, proteins translated by ribosomes are folded and secreted in a folded state. In addition, it does not require the action of a chaperone or the like, is transported to the cell membrane only by the action of the signal, is secreted through the membrane protein TatA-D, and the signal and the protein are separated by the action of SPase (Fernando A. Agriberres, J. Fred Dice; Protein translocation cross members [see Biochimica et Biophysica Acta 1513, 1-24.2001].

Advantages of Actinomycete Secretion Expression System This secretory mechanism can also be used when a foreign gene is expressed by gene manipulation to produce a protein. When a heterologous protein is produced only in the cell, if it is produced in a very small amount, it is likely to be degraded by intracellular protease, and conversely, even if it is produced in a large amount, the loss due to degradation, Depending on the state of the synthesis field, it may not become an active mature protein, or production may reach a peak. In fact, when a heterologous protein is produced in E. coli cells by genetic manipulation, it can be up to about 20% of the protein in the cell, but often becomes an insoluble mass. To normalize this, a redox agent such as glutathione is used, and a troublesome operation of “unwinding” is required. In addition, even in the case of secretion, Gram-negative bacteria such as Escherichia coli have double cell membranes, and the target protein is accumulated in the space (periplasm) of the two layers of membranes, which requires an extraction operation. Gram-positive bacteria such as actinomycetes have only a single cell membrane, so the protein is completely out of the cells and accumulates in the culture medium, so mass production is expected and extraction from the cells is simplified (See Hiroshi Matsuzawa, Basics of Protein Engineering, Tokyo Kagaku Doujin (2004)).

<Currently recombinant expression system>
Recombinant expression of proteins can vary from transient, small-scale expression for identification and confirmation of genes and cDNAs to large-scale expression at the factory level for protein isolation. There is. There are various types of proteins used as hosts for protein expression. Escherichia coli is most frequently used because of its simplicity and historical background, but bacteria such as Bacillus subtilis and actinomycetes, yeast, mammalian cultured cells, insects and their cultured cells, plant cells, and molds have been reported. These expression systems have their own characteristics, and it is necessary to select an optimal system that suits the purpose after fully understanding the characteristics of each expression system.

  The expression system in E. coli is rich in traditional knowledge, can be applied to the expression techniques accumulated so far, is easy to culture and low in cost, and is the first system considered as a host for mass expression. Proteins expressed in E. coli cells are usually accumulated as inclusion bodies (insoluble proteins that do not have the correct three-dimensional structure). This is caused by the fact that the amount of expressed protein in Escherichia coli is so large that it is not correctly folded, which is a major obstacle to protein function research. In order to adjust the inclusion body to an active protein, it is necessary to solubilize the insoluble matter with a denaturing agent such as urea (urea) or guanidine hydrochloride and then regenerate it from the denaturation. Alternatively, a plurality of molecular chaperones known to cooperate in protein folding can be cloned using vectors encoded on a single plasmid. One of the major problems with the E. coli expression system is that various modifications including the addition of sugar chains cannot be made.

  Yeast is often used because it is as easy to culture as E. coli. There are a case where it is expressed in the microbial cell and a case where it is expressed secreted outside the microbial cell with a signal peptide attached. Sugar chains can be added, but complex sugar chains found in animal cells cannot be added. In addition, like E. coli, there is a drawback that modification such as acylation and amidation cannot be performed.

  Animal cells are used for expression that requires post-translational modifications to proteins, but culture requires expensive serum, long culture time, and low protein production. As described above, animal cells have many aspects that are not suitable for mass production, and their use is limited compared to E. coli for the purpose of producing pharmaceuticals or for small-scale expression at the laboratory level. Insect cell culture requires serum, but since it can be cultured in a closed system, the culture apparatus becomes simple and the productivity is relatively high. In recombinant expression in eukaryotic cell systems such as animal / insect cells and yeast, the expressed protein is often soluble, and in that case no solubilization or denaturation recovery operations are required (Table 2, Non-Patent Document 4 and Jiro Ota). , Introduction to Microbiology-Biotechnology (see Asakura Shoten (1992)).

<About GFP>
GFP (Green Fluorescent Protein) is a protein that shifts the blue light (maximum 470 nm) of the chemiluminescent protein, Aequorin (eocrine) to green light (maximum 508 nm) in Aequorea victoria (1962). It was discovered by Dr. et al., And its cDNA was cloned by Prascher et al. The primary structure (amino acid sequence) of GFP is a protein consisting of 238 amino acids, and the chromophore p-hydroxybenzylideneimidazoline is formed from the 65th to 67th amino acids serine, tyrosine and glycine. As research on the luminescence mechanism of fluorescent proteins of GFP progressed, mutants of GFP proteins were created by Heim, Delagrave, Crameri, and Cormach, and the usefulness of these fluorescent proteins has been reported. Among them, the S65T mutant in which the 65th serine is replaced with threonine shifts to a long wavelength side with an excitation maximum wavelength of 490 nm and emits fluorescence several times stronger than wtGFP. It has been reported that it can impart properties such as promoting protein solubility and increasing protein solubility. Currently, various mutant GFPs are commercially available. CLONTEC's Enhanced Green Fluorescent Protein (EGFP) is optimized for the human codon usage in addition to chromophore amino acid substitution (P64L, S65T, etc.). The above mutations have been introduced. Furthermore, the fluorescent color variants EBPF (Blue) (Y66H amino acid substitution, etc.), ECFP (Cyan) (Y66W amino acid substitution, etc.), and EYFP (Yellow) (T203Y amino acid substitution, etc.) have been used by the company. It can be obtained as a mutant that can be observed with an emission color different from that of EGFP. The company has also created destabilized mutants and mutants with shifted excitation wavelength, increased solubility, and reduced cell toxicity (Satoshi Miyawaki, GFP and Bioimaging Yotei ( 2002)).

  There are three main types of expression when using GFP. There are three types: single expression of GFP, addition of localization signal to GFP, and fusion of GFP and functional protein.

<About cytochrome P450>
Cytochrome P450 (Cytochrome P450, hereinafter referred to as P450) is a group of heme proteins widely distributed in the living world from microorganisms to plants and animals, and exhibits a characteristic absorption spectrum having a maximum at 450 nm by carbon monoxide bonding in a reduced form. The existence of an extremely large number of molecular species with monooxygenase-type oxygenase activity and different substrate specificities of the reaction to be catalyzed is known. It is concluded that it is a gene family that is differentiated and diversified from the common ancestral gene in the process of biological evolution.

  The 450 nm absorption spectrum of the carbon monoxide conjugate that is characteristic of P450 is that the thiolate anion (-S-) in which the -SH group of the cysteine residue of P450 protein is ionized is coordinated to the fifth coordination site of protoheme. Because it is. The amino acid sequences before and after this cysteine residue are well conserved for all P450s and are called “heme binding regions”. In the protoheme protein whose absorption spectrum maximum of a carbon monoxide conjugate such as hemoglobin is at 420 nm, the fifth ligand is imidazole of the histidine residue. In P420, which is a modified form of P450, the fifth ligand is replaced by imidazole due to the structural change of the protein molecule, and the absorption spectrum of the carbon monoxide conjugate is almost the same as that of hemoglobin.

  The basic molecular structure of P450 was revealed by X-ray crystallography. The P450 molecule is a prism type with a side of about 60 mm and a thickness of about 30 mm, and is characterized by a long I helix that penetrates the entire molecule. Inside the molecule is a space where hydrophobic substrate molecules can be accommodated and fixed, and the hem that activates oxygen molecules is located at the bottom of the molecule, so it is called a hem pocket.

Most P450s function as monooxygenases, but this reaction requires the provision of reducing power to reduce heme iron and activate oxygen molecules bound to P450 together with the presence of molecular oxygen. This P450 reduction system has the following four types.
(1) NADPH-cytochrome P450 reductase (P450 reductase)
(2) Cytochrome b5-NADH-cytochrome b5 reductase system (b5 system)
(3) NAD (P) H-iron-sulfur protein system (ISP system)
(4) Fusion protein of P450 and reduction system (fusion protein system)
(1) and (2) are found in the endoplasmic reticulum of eukaryotic cells, and (3) are found in mitochondria and bacteria. (4) is found in bacteria and molds, but there are still few examples. P450 may be classified by its reduction system. Those that receive electrons from the ISP system are called class I, those using P450 reductase are called class II, and those obtained by fusing P450 with a reductive system having the same composition as P450 reductase are called class III.

  The research of P450 is recognized as important in the fields of medicine, pharmacy, and agriculture, and attempts have been made to apply P450 that catalyzes a highly specific oxygenation reaction to industrial production of organic compounds. It seems possible to modify the substrate binding site of the P450 molecule to create P450 with any substrate specificity, and the use of P450 for industrial production is expected to be one of the new development directions (Omura Tsuneo, P450 Molecular Biology (see Kodansha (2004)).

<CYP102A3>
P450BM3 (CYP102A1) discovered in Bacillus megaterium has a unique structure in which P450 and P450 reductase are fused to exist as a single polypeptide chain. It is a reaction fatty acid subterminal (ω-1 to ω-3) hydroxylation reaction to be catalyzed. It also epoxidizes unsaturated fatty acids. As a result of genome analysis, the presence of two types of homologues (CYP102A2 and CYP102A3) has also been clarified in Bacillus subtilis. Therefore, this type of P450 may exist universally in bacteria of the genus Bacillus. Since fatty acids are toxic to Bacillus subtilis, this P450 is considered to be meaningful as a detoxifying enzyme for Bacillus subtilis (Omura Tsuneo, P450 Molecular Biology Kodansha (2004), Andrew W. Munro, Gordon Lindsay; Bacterial Cytochromes P) -450 [Molecular Microbiology 20 (6), 1151-1125.1996] and Andrew W. Munro, Kirsty J. McLean; Cytochrome P450-redox partner fusion enzymes [Biochimicaet 6].

<CYP106A3>
CYP106A2, discovered from Bacillus megaterium ATCC 13368, catalyzes a substrate of the steroid skeleton. It is used in the field of biotechnology to carry out a reaction of hydroxylating important steroids such as progesterone and 11-deoxycortisol (C. Virus, R. Bernhardt; Function and engineering of the 15β-hydroxylase Cyt106A2cCyVaciolCytoCycciAt. .34 (6), 1215-1218.2006].

<About hemoglobin>
Hemoglobin is a protein in red blood cells that are present in the blood. It has the property of binding to oxygen molecules and is responsible for transporting oxygen from the lungs to the whole body. It has a red color due to the presence of iron ions. Each subunit is composed of a polypeptide portion called globin and one heme portion, and hemoglobin consisting of four types of globin was used in the present example as described later.

  As mentioned above, although embodiment of this invention was described with reference to drawings, these are the illustrations of this invention, Various structures other than the above are also employable.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these.

<Overview>
In this embodiment, high activity mutant PLD (C.Ogino acquired in an expression system of E. coli, H.Fukuda; Phosoholipase D from Streptoverticillium cinnamoneum: protein engineering and application for phospholipids production (Journal of Molecular Catalysis B: Enzymatic 23, 107-115.2003)) was constructed and analyzed to construct an expression system that can be secreted in large quantities by actinomycetes. In addition, for the application to the secretory production system of useful proteins using actinomycetes as a host, we first constructed several foreign protein expression systems as model proteins, and this actinomycetes expression system is effective for the production of foreign proteins. We examined whether it was.

  For this purpose, the present inventors have investigated the applicability of this recombinant expression system to various protein expression systems, and various proteins in the PLD gene region of the construction plasmid (pUC702-promoter-PLD). We developed a host vector system that incorporates the gene and enables the secretory expression of foreign-derived proteins. The inventors succeeded in expressing eukaryotic heme-containing proteins and Bacillus subtilis-derived heme-containing proteins.

  Referring to FIG. 1 showing this scheme, PCR primers are designed so as to have a NheI site on the 5 ′ side of the target gene and a BamHI or BglII sequence on the 3 ′ side to amplify the target gene. Thus, it was possible to insert the gene into this vector (pUC18 + promoter + term). This scheme shows a scheme for inserting a green fluorescent protein (GFP) gene as a model system.

  Since the inserted plasmid (pUC18-pro-gfp) itself is a vector that is difficult to insert (transform) into actinomycetes, a shuttle vector that can be transformed with both actinomycetes and E. coli. The gene was inserted into. From the Promoter region inserted into the plasmid (pUC18-pro-gfp) to the end of the Terminator region, it was possible to remove them with restriction enzymes HindIII and KpnI. By inserting this excised sequence into the actinomycetes / Escherichia coli shuttle vector pUC702, the construction of a vector capable of secretory production in large quantities by actinomycetes was completed. The expression of eukaryotic heme-containing protein and Bacillus subtilis-derived heme-containing protein could be confirmed using this vector system.

  The results of this Example will be described in more detail. T.A. -It was found that mass secretion can be expressed in the same manner as PLD. Furthermore, by conducting a three-dimensional structure analysis of the mutant PLD, W.M. T.A. It was speculated that only the lid-like structure (hereinafter referred to as Lid region) existing in the vicinity of the active site was changed as compared with -PLD. As a result, it was shown that specific activity, substrate affinity and catalytic ability changed. Moreover, it was found that there was no relationship with the expression system of E. coli, and it was shown that a mutant having low activity in the expression system of E. coli may have higher activity in the expression system of actinomycetes. Furthermore, in order to apply this expression system to the expression of various foreign-derived proteins, a general-purpose vector was developed, and secretion expression of the foreign protein was confirmed in the expression system of actinomycetes.

<Experimental materials and equipment>
[Reagent used]
DNA related・ Nacal nacalai tesque
・ Tripton nacalai quest
・ Yeast extract nacalai tesque
・ Agar Powder (agar powder) nacalai tesque
・ Agarose LE (low electroosmosis) classic type [for electrophoresis]
nacalai tesque
・ SDS nacalai quest
・ KOAc nacalai tesque
・ Acetic acid nacalai tesque
・ Chloroform nacalai tesque
・ 2-propanol (isopropyl alcohol) nacalai tesque
・ Sodium acetate Kanto Chemical Co., Ltd. ・ Ethanol nacalai tesque
・ Methanol nacalai tesque
・ Ethidium bromide nacalai tesque
・ Tris nacalai tesque
・ HCl Wako
・ EDTA nacalai quest
・ Glycerol nacalai tesque
・ Bromophenol Blue (BPB)
nacalai tesque
・ Dimethyl Sulfoxide (DMSO)
Wako (046-21981
Lot. TCQ2620)
・ 6 × Loading Dye TOYOBO
・ Molecular weight marker (λ / EcoRI / HindIII)
TOYOBO, Wako
・ Ligation high TOYOBO
・ MagExtractor, Gel purification kit
TOYOBO NPK-601
・ Restriction enzyme
Alw44I, BamHI, BglII, EcoRI, HindIII,
KpnI, NheI, PstI, SphI, XbaI
TOYOBO
・ DNA Polymerase
Pyrobest, PrimeSTARTM HS
TaKaRa
KOD-Plus TOYOBO
・ BigDye ver1.1, BigDye ver3.1, Hi-Di Formatide, POP-6 Polymer, Buffer 10 × with EDTA
Applied
Biosystems

Protein-related / APS nacalai tesque
・ 2-Mercaptoethanol nacalai tesque
・ N, N, N ′, N ′,-Tetramethylethylenediamine (TEMED)
nacalai tesque
Silver staining II kit Wako (291-50301)
・ Na ₂ HPO ₄ nacalai quest
・ NaH ₂ PO ₄ nacalai tesque
・ Tween 20 (Polyoxyethylene Sorbitan Monolaurate nacalai quest
356-24
・ Skim milk nacalai tesque
Anti-GFP Epitope Tag Antibody (Rabbit Polyclonal IgG)
Affinity
BioReagents
(PA1-980A)
・ Anti-Rabbit IgG (FC), AP Conjugate
Promega (S3731)
BCIP / NBT Color Development Substrate
Promega (S3771)

Actinomycete culture-related K ₂ SO ₄ nacalai tesque
・ KH ₂ PO ₄ Wako
・ MgCl ₂ Wako
・ CaCl ₂ · 2H ₂ O nacalai tesque
・ Glucose nacalai tesque
・ Sucrose nacalai tesque
・ Glycine nacalai tesque
・ TES (N-Tris (hydroxymethyl) methyl-2-aminoethanesulfonic Acid)
nacalai tesque
· Tryptic Soy Broth (TSB) <The composition per 30 g / l is shown below> BECTON DICKINSON
Pancreatic Digest of Casein (17.0 g / l)
Enzymatic Digest of Soybean Meal
(3.0 g / l)
Dextrose (2.5 g / l)
Sodium Chloride (5.0 g / l)
Dipotassium Phosphate (2.5g / l)
・ BACOTTM Peptone BECTON DICKINSON
・ Yeast extract BECTON DICKINSON
・ CASAMINO ACIDS BECTON DICKINSON
・ MALT EXTRACT BECTON DICKINSON
・ Lysozyme, from Egg white
Wako (126-02671)
・ Thiostrepton Streptomyces azureus, SIGMA (T8902-1G)
・ Polyethylene glycol (PEG) 1,000
Fluka (81188)
・ 5-Aminolevulinic acid hydrochloride
Cosmo Bio (AL-00-2)

[Device used]
・ Deep freezer (-85 ℃)
ULTRA LOW MDF-192 (SANYO)
・ Autoclave HIGH-PRESSURE STEAM STERILIZER BS-305 (TOMY)
KT-2346 (ALP)
・ Bio Shaker BIO SHAKER BR-40LF (TAITEC)
Bio Shaker BR-41FL (TAITEC)
・ PERSONAL-11 SD set (TAITEC)
Program Incubator IQ802 (yamato)
EYELATRON FLI-301N (EYELA)
・ Incubator SOFT INCUBATOR SLI-450N (EYELA)
INCUBATOR MIR-153 (SANYO)
COOL INCUBATOR PCI-301 (ASONE)
・ Clean Bench BIO-LABO CLEANBENCH NS-8A (Juji Field)
Bio Clean Bench VCB-1600S (Oriental)
・ Centrifuge Inverter ・ Compact high-speed cooling centrifuge 6930 (KUBOTA)
Microtec 1524R (ASTEC)
H-103n (KOKUSAN)
himac CT13 (HITACHI)
Centrifuge 5415 R (eppendorf)
MR-150 (TOMY)
CAPSULEFUGE PMC-060 (TOMY) (tabletop centrifuge)
・ Spectrophotometer Spectrophotometer U-3010 (HITACHI)
・ Agarose electrophoresis device Mupid-2plus (ADVANCE)
・ PAGE electrophoresis apparatus POWER STATION 1000VC AE-8270 (ATTO)
POWER STATION 1000VC AE-8450 (ATTO)
・ UV sample photographing device FAS-III (TOYOBO)
DIGITAL IMAGE STOCKER DS-30 (TOYOBO)
・ Block incubator BLOCK INCUBATOR BI-515 (ASTEC)
BLOCK INCUBATOR BI-516C (ASTEC)
D-THERMO 501 (APEL)
HEATING BLOCK HF-41 (YAMATO)
・ Vacuum drying equipment VC-36N (TAITEC)
VC-12S (TAITEC)
・ Vortex mixer
TUBE MIXER TM-2000 (IWAKI)
VORTEX-GENIE2 G-560 (Scientific Industries)
PCR apparatus PCR System 2700 (Applied Biosystems)
PCR System 2720 (Applied Biosystems
Electronic balance BJ 610 (Sartorius)
AX 200 (SHIMADZU)
・ Distilled water equipment STILL ACE SA-2000E (EYELA)
DNA sequencer ABI PRISMTM 310 Genetic Analyzer (Applied Biosystems)
-Electroporation system Gene Pulser XcellTM electroporation system (BIO-RAD)
・ Microscope ECLIPSE 50i (Nikon)
Birkelturk hemocytometer (Elma Sales Co., Ltd.)
・ Ion exchange chromatograph GRADENT PNMP
ECONO UV MONITOR
MODEL 2110 FRACTION COLLECTOR

[Bacteria]
・Escherichia coli (E. coli)
Novablue (Novagene)
DH5-α (TaKaRaBio)

· Actinomycetes (Streptomyces)
Streptomyces lividans 1326 strain Culture temperature; 28 ° C., medium; TSB.
Actinomycetes used when Fukumoto et al. Started this experiment. The source is a strain distributed from the National Food Research Institute under the jurisdiction of the Ministry of Agriculture, Forestry and Fisheries (commonly known as AIST), via Prof. Shinji Tokuyama, Department of Agricultural Chemistry, Faculty of Agriculture, Shizuoka University.

Streptomyces lividans (also known as Streptomyces violaceoruber) (NBRC 13385)
Culture temperature; 28 oC, medium; TSB.
In 2005, the strain was purchased from the Biogenetics Resources Division (NBRC), Biotechnology Headquarters, National Institute of Technology and Evaluation, transferred from the Fermentation Research Institute (IFO, Institute of Fermentation, Osaka). The genetic property is equivalent to that of the above Streptomyces lividans 1326 strain and can be applied to transformation of pIJ702 series plasmids.

Streptovicillium cinnamoneum (also known as Streptomyces cinnamoneus) (IFO12852, now NBRC12852)
Culture temperature: 28 ° C., medium: TSB.
This strain was screened by Hideki Fukuda and others at Kobe University as a production strain that secreted and produced a large amount of PLD having high phosphorylation activity in the medium. Sugano et al. Determined the PLD gene sequence derived from this strain and became the genetic material used in the gene recombination system with the pIJ702 series plasmid. It has also been clarified that an expression promoter that acts very strongly on secretory production is present upstream of the PLD gene of this strain. In this sense, it is the original strain of this study.

  [Sample composition]

[Primers used]
For DNA fragment amplification
Promoter region amplification S-HindIII-promoter (SEQ ID NO: 7)
5'-CCCAAGCTTACGTCATGGCGGGTCTCTCTCGTC-3 '
As-BamHI-NheI-signal (SEQ ID NO: 8)
5'-CCCGGATCCGCTAGCGAAGGCCGGAGCCGCGGGCAG-3 '

Terminator region amplification S-BamHIII-BalII-Terminator (SEQ ID NO: 9)
5'-CCGGATCCAGATCTTGAGACGACTGAGCGCCCGGA-3 '
As-EcoRI-kpnI-Terminator (SEQ ID NO: 10)
5'-CCGAATTCGGTACCATTTCCTCGCTGGTCGGTTCG-3 '

NheI-GFP for amplification of GFP and BFP genes (SEQ ID NO: 11)
5'-CCGCTAGCATGGCCAGTAAAGGAGAAGAACTCTTCACTGGA-3 '
As-BglII-BamHI-GFP (SEQ ID NO: 12)
5'-CCAGATCTGGATCCTCAGTTGTACAGTTCATCCATGCCATG-3 '

XbaI-A1 for A1 gene amplification (SEQ ID NO: 13)
5'-CCCTCTAGAGTATGTAACCGACTTGAGCAG-3 '
As-BamHI-A1 (SEQ ID NO: 14)
5'-CCCGGATCCTTAGCCGGAAATACCGCTAGC-3 '

NheI-A2 for A2 gene amplification (SEQ ID NO: 15)
5'-CCCGCTAGCGATTGCACTTCCCTCAATCGC-3 '
As-BamHI-A2 (SEQ ID NO: 16)
5'-CCCGGATCCTTAACCAGAAATGCCGCTGAC-3 '
S-repair-A2 (SEQ ID NO: 17)
5'-GTTGGAGCTCGTAGCTTCGACAAC-3 '
As-repair-A2 (SEQ ID NO: 18)
5'-GTTGTCGAAGCTACGAGCTCCAAC-3 '

NheI-B1 for B1 gene amplification (SEQ ID NO: 19)
5'-CCCGCTAGCGAATGCTGCAGTAGAGGTGAT-3 '
As-BglII-B1 (SEQ ID NO: 20)
5'-CCCAGATCTTTACAAGCCTGCACCAATACC-3 '

NheI-B2 for B2 gene amplification (SEQ ID NO: 21)
5'-CCCGCTAGCTCGAGCTGTTGTTCCTCCGAG-3 '
As-BamHI-B2 (SEQ ID NO: 22)
5'-CCCGGATCCTTACAAGCCTGCTGAGATGCC-3 '

S-XbaI-CYP106 for CYP106A2 gene amplification (SEQ ID NO: 23)
5'-CCCTCTAGAATGAAAGAAGTTATTGCAGTA-3 '
As-BglII-CYP106 (SEQ ID NO: 24)
5'-CCCAGATCTTTACATGCGGCTTGCCTTAAG-3 '
S-repair-CYP106 (SEQ ID NO: 25)
5'-GATGATATCATCTCTGATCTATTGA-3 '
As-repair-CYP106 (SEQ ID NO: 26)
5'-GACTTCAATAGATCAGAGATGATAT-3 '

S-NheI-CYP102 for CYP102A3 gene amplification (SEQ ID NO: 27)
5'-CCCGCTAGCATGAAACAGGCAAGCGCAATA-3 '
As-BglII-CYP102 (SEQ ID NO: 28)
5'-CCCAGATCTCTACATTCCTGTCCAAACGTCTTT-3 '

For sequence
S-M13Fw primer (SEQ ID NO: 29) for confirmation of promoter region upon incorporation into pUC18
5'-GGGTAACGCCAGGGTTTTCCCAG-3 '

In pUC18, a terminator is used. In pUC702, an AS-M13Rv primer (SEQ ID NO: 30) is used for promoter region confirmation.
5'-GGATAACAATTTCACACAGG-3 '

When incorporating into pUC702, Rv-pUC702 (SEQ ID NO: 31) for terminator region confirmation
5'-CCCGGGAGTAATCCTGGGATTAC-3 '

Promoter region, Promoter -1 for confirmation of Signal region (SEQ ID NO: 32)
5'-GCGCGGCGCCCCGCGGCTCGT-3 '
As-promoter-2 (SEQ ID NO: 33)
5'-ACCCTCGCCCGGCAGCGCGGT-3 '
promoter-3 (SEQ ID NO: 34)
5'-TAGCCGCGGGCCACGACGTCC-3 '
Fw-promoter (SEQ ID NO: 35)
5'-CGTGCGCCGACTCCACGTCCGTGCC-3 '
Fw-rbs (SEQ ID NO: 36)
5'-CCCTCTCGGAGGCGGCCTGCCGTACC-3 '

Gfp-1 for GFP gene confirmation (SEQ ID NO: 37)
5'-CCATACGGAAAACTTACCCTGAA-3 '

P450-106A2 (Seq1) for CYP106A2 gene confirmation (SEQ ID NO: 38)
5'-GGTGGATACCTTATTTCTTC-3 '

P450-102A3 (Seq300) for CYP102A3 gene confirmation (SEQ ID NO: 39)
5'-GCCCACCGCATTTTGCTGCC-3 '
P450-102A3 (Seq600) (SEQ ID NO: 40)
5'-GAAAACGAAGCTGCAGTTCC-3 '
P450-102A3 (Seq900) (SEQ ID NO: 41)
5'-GTTAACGGATGACACGCCTG-3 '
P450-102A3 (Seq1200) (SEQ ID NO: 42)
5'-GCGCTTGTATTGGCATGCAG-3 '
P450-102A3 (Seq1500) (SEQ ID NO: 43)
5'-GGTGAACTGGCTGCTCAAGG-3 '
P450-102A3 (Seq1800) (SEQ ID NO: 44)
5'-CAGCGATTGGGGAAGGTGAC-3 '
P450-102A3 (Seq2100) (SEQ ID NO: 45)
5'-CATATCGGAATCCTGCCAAAG-3 '
P450-102A3 (Seq2400) (SEQ ID NO: 46)
5'-CAAAACGTCTTACCATGCTTG-3 '
P450-102A3 (Seq2700) (SEQ ID NO: 47)
5'-CGCCTATGAT

<Experiment method>
[Basic operations in genes]
Currently, the PCR (Polymerase chain reaction) method is generally used for amplification of the target DNA. The PCR method is an epoch-making method that can specifically amplify only the target sequence in a few hours more than 1 million times using a very small amount of DNA as a template. At present, it is applied in various fields ranging from simple operation to basic research, clinical examination, and criminal investigation.

DNA amplification (PCR)
The PCR method is a method of amplifying a target DNA fragment exponentially in a test tube, and is a technique that has been made possible for the first time by using a heat-resistant DNA polymerase (DNA polymerase). In the PCR reaction, DNA is amplified by repeating three steps as one cycle. What is necessary for this reaction is a DNA as a template, a short DNA fragment (Primer) corresponding to the 5 ′ end of the gene portion to be amplified, a short DNA fragment (Primer), deoxyribonucleotides, a salt and a heat-resistant DNA polymerase It is.

  By applying heat at around 95 ° C. in the first step, the double-stranded template DNA is made into a single-stranded state (Denature), and the primer is allowed to bind to the set sequence portion in the template DNA. . Next, by lowering the temperature, the primer binds to the complementary sequence portion of the template and partially forms a double-stranded state (annealing). Finally, a thermostable polymerase synthesizes DNA in the 5 '→ 3' direction starting from the primer bound to the template, and extends the DNA.

  By repeating the simple cycle as described above, it becomes possible to amplify the target DNA fragment, but each condition is not uniquely determined, and the optimum condition is determined according to the purpose. There is a need to.

Items to be considered in the PCR method As mentioned above, the cycle in PCR is basically composed of three stages: heat denaturation, annealing, and extension reaction, of which the annealing temperature must be set independently, and The time taken for each step and the number of cycles.

  The annealing temperature is usually determined with reference to the Tm of each primer. When the Tm of the two primers is greatly different, it is set near the lower Tm. First, follow the instructions for the polymerase for the time and cycle of the steps. In addition, Extension Time is performed at an amplified fragment of 1 min / 1 kbp.

Agarose Gel Electrophoresis
In the case of double-stranded DNA, the base charges cancel each other out by hydrogen bonds between complementary strands, so that the entire molecule is mainly the negative charge of the phosphate group. Further, since the number (charge) of phosphate groups is proportional to the number of nucleotides (molecular weight of DNA), all DNA molecules are attracted to the anode with a constant force per mass. Furthermore, DNA having a double helix structure has the same linear molecular shape regardless of the base sequence, and hardly affects the mobility of the three-dimensional structure. That is, the size (length) of the molecule has the only influence on the mobility. The longer the molecular weight, the slower the migration mobility. DNA is separated using a principle that depends only on the length of the DNA strand.

1) Agarose is weighed and placed in an Erlenmeyer flask, 1 × TAE is added to a microwave oven, and agarose is dissolved.
2) When the solution in which agarose is dissolved and becomes homogeneous is cooled to around 50 ° C., add ethidium bromide solution (10 mg / ml) at a rate of 5 μl per 100 ml of gel and mix well.
3) Pour the gel into the gel molding tray and prick the comb.
4) Let the gel stand at room temperature for about 30 minutes, and when it cools and hardens sufficiently, remove the comb and set the gel in the electrophoresis tank.
5) Fill the electrophoresis buffer (1 × TAE) to the top of the gel.
6) Add 1/5 volume of 6 × loading dye to the sample DNA.
7) Gently place the sample into the gel hole.
8) Place Marker 2 in the gel hole.
Marker2 is a double digestion of λ (d857Sam7) with restriction enzymes HindIII and EcoRI.
It is used as a DNA molecular weight marker in the high to medium molecular region.
9) When BPB (bromophenol blue) flows about 1/2 or 2/3 of the gel (about 20 min), the electrophoresis is stopped.
10) Irradiate UV with a transilluminator (wavelength 302 nm).

Gel purification
This is a method for recovering only a DNA fragment of a desired size after PCR, Restriction Enzyme Digestion or the like.

1) Agarose electrophoresis, ethidium bromide staining.
2) A target band is cut out with a scalpel so as to be as small as possible under UV irradiation (Long wave).
3) Finely slice the cut gel and transfer it to a 1.5 ml tube.
4) Add 400 μl adsorbate and leave with occasional stirring until the gel is completely dissolved. * If it is difficult to melt, leave it at 55 ° C.
5) Add 30 μl of magnetic beads and let stand for 2 min with occasional vortexing.
6) Separate B / F, add 600 μl of washing solution and vortex for 10 sec.
7) Separate B / F, add 1 ml 75% ethanol and vortex for 10 sec.
8) Separate B / F, spin down, completely remove supernatant.
* Open the lid of the tube and leave it at 55 ° C for 5 min.
9) 25-100 μl of T.I. E. And vortex for 10 sec.
10) Let stand for 2 min, separate B / F and collect supernatant.

Alcohol Precipitation
In the simplest method used to purify and concentrate nucleic acids, DNA, which is a polymer colloid, is condensed with alcohol and salt to obtain a precipitate. Depending on the type of alcohol used, there are ethanol precipitation and isopropyl alcohol precipitation. Since excess salts and low-molecular components do not precipitate, DNA concentration and buffer replacement can be easily performed. In the case of ethanol precipitation, RNA is also co-precipitated, but when DNA is precipitated using polyethylene glycol (PEG), which is a polymer alcohol (PEG precipitation), RNA co-precipitation hardly occurs. PEG precipitation is often used when RNA needs to be removed, such as

Ethanol Precipitation
This is the most common method of alcohol precipitation. The yield of DNA increases in proportion to the cooling time.

1) About 1/10 times the amount of 3M NaOAc in Sample (it may not be added if Sample contains salt as in Alkali I-II-III) and 2-2.5 times the amount of low temperature 100 Add% Ethanol and Voltex.
2) Cool at −20 ° C. for 1 h or at −85 ° C. for 20 min or longer, and centrifuge at 4 ° C., 13000 rpm for 30 min to precipitate DNA.
3) Carefully remove the supernatant, add 70% Ethanol, voltex, centrifuge at 4 ° C, 13000 rpm for 5 min and wash. (There is also a cleaning method in which 70% Ethanol is poured into the tube wall surface only twice.)
4) Dry completely with a vacuum dryer.
5) Add an appropriate amount of TE or DW and dissolve.

Isopropanol Precipitation
Since a small amount of alcohol may be added, it is suitable for a large sample amount. Since the yield does not change even after cooling for 1 hour or longer, a shorter time is required than ethanol precipitation.

1) Add about 1/10 times the amount of 3M NaOAc to Sample (as in the case of alcohol precipitation, it may not be added if Sample contains salt) and add 100% Isopropanol and Voltex.
2) Hereinafter, similar to ethanol precipitation.

PEG Precipitation
Polyethylene glycol (PEG), which is a polymer alcohol, promotes aggregation and precipitates DNA molecules by depriving DNA of hydration water. * Use to remove RNA.

1) Add an equal amount of PEG6000 Solution to Sample and Voltex.
2) Cool at 4 ° C. for 1 h.
3) Centrifuge at 4 ° C. and 13000 rpm for 30 minutes to precipitate DNA.
4) Hereinafter, similar to ethanol precipitation.

Restriction Enzyme Digestion
A type of restriction enzyme or endonuclease that recognizes a specific sequence of DNA and cleaves that site. By utilizing this property, it becomes possible to cut out only the target sequence from the sequence of an arbitrary DNA or to incorporate it into another DNA. Further, it is used to confirm whether or not the target DNA sequence is incorporated at an accurate position.

1) When Sample is in the form of a plasmid, heat at 98 ° C. for 3 minutes and cool on ice.
2) Sample restriction enzyme (within 5% of total reaction volume), 10 × Buffer, optional D.E. W. Add
3) React at 37 ° C. for 3 to 6 hours.

Ligation
In this method, Vector linearized by restriction enzyme treatment and DNA fragment are linked by phosphodiester.

1) A DNA fragment to be incorporated and a vector are mixed at (3: 1 to 50: 1) according to the DNA concentration to prepare a sample.
2) A half amount to an equal amount of Ligation High (TOYOBO) is mixed with Sample.
3) React at 16 ° C. for 6 h to overnight.

Competent Cell
When performing gene transformation using E. coli as a host, the cell wall is made competent so that E. coli can easily take up the target plasmid DNA. Competent cells can be frozen with glycerol at −80 ° C. to preserve viability and can be stored for several years. This method is optimized for the DH-1 strain, but is applicable to most E. coli. By this method, competent E. coli Nova Blue, DH-5α strain is also prepared.

All work should be done in a clean bench, paying attention to contamination.
1) Using a spreader sterilized with 70% Ethanol, uniformly spread 50 μl of E. coli to be made competent on an LB plate (without antibiotics).
2) Incubate overnight at 37 ° C.
3) E. coli 1loop is inoculated into 50 ml of LB medium (no antibiotics) or Tf medium, and further cultured overnight at 37 ° C. (Mini culture).
4) Add 20 ml of the culture solution to 400 ml of LB medium (without antibiotics), and culture at 37 ° C. for about 3 h until OD600 = 0.5 (Large culture).
5) Cool on ice for 5 min.
6) Centrifuge for 15 min at 4 ° C. and 4000 × g, and remove the supernatant.
7) Add 80 ml of ice-cooled Tfb-1 to the collected cells and suspend by pipetting.
8) As in 5-7, cool and centrifuge for 5 min, remove the supernatant, add 16 ml of Tfb-2 and suspend by pipetting.
9) After cooling on ice for 15 min, quickly dispense into 200 μl aliquots.
10) Store in a deep freezer (-85 ° C).

Transformation
Transformation is a method for introducing new genetic information into E. coli or cultured cells using an appropriate vector such as plasmid. To date, transformation systems using plasmids with a broader range of applications have been studied, and at present, transformation in E. coli systems is the most widely used method. In this study, E. coli DH5-α strain was used for gene cloning.

1) Add 100 μl of competent cell to 2.0 μl of Plasmid (5 μl after ligation), and cool on ice for 30 min.
2) Heat-treat for 45 seconds at 42 ° C. and cool on ice for 5 minutes or more.
3) Add 1 ml of LB solution (without Amp) and incubate at 37 ° C. for 1 h.
4) Centrifuge at 3000 rpm for 10 min and remove the supernatant.
5) Suspend the collected cells by pipetting and spread the transformant of E. coli uniformly on the LB plate (with Amp) using a spreader sterilized with 70% Ethanol.
6) Incubate at 37 ° C overnight.

Alkali-SDS
Escherichia coli serving as a host for plasmid amplification contains genomic DNA and various RNAs as DNA other than plasmids, and proteins and lipids as constituents of the cells. The alkaline method is a method for purifying plasmid DNA from these mixtures.

Procedure 1) Pick up single colonies with autoclaved toothpicks, inoculate into 3 ml of LB medium (with Amp), and culture overnight at 37 ° C.
2) Remove the toothpick with forceps and centrifuge at 3000 rpm for 10 min to precipitate the bacteria, and discard the supernatant.
3) Add 100 μl of Solution I and vortex to suspend the bacteria.
4) Add 200 μl of Solution II (100 μl of 4N sodium hydroxide, 100 μl of 1% SDS) and gently stir.
5) Add 200 μl of Solution III, mix gently, and transfer to a tube.
6) Add 50 μl of chloroform and vortex, then centrifuge at 4 ° C., 13000 rpm for 10 min.
7) Collect the upper layer (aqueous phase) while taking care not to allow precipitation (the white precipitate present at the boundary is a denatured protein and prevents contamination as much as possible).

RNase treatment
When DNA is extracted from cells during plasmid extraction, RNA present in large amounts in the cells is contaminated. Therefore, when RNA contamination has an adverse effect, such as a Sequence reaction, it is necessary to remove the RNA, so an RNase treatment is performed to decompose the RNA.

1) Add about 10/500 times the amount of R10 to Sample.
2) Leave at 37 ° C. for 30 min to 6 h.

Alkali Denaturation
In this method, double-stranded DNA is denatured into single strands by alkali. By this method, DNA is easily nicked, the nicked DNA strand becomes a linear DNA by alkali denaturation, and the DNA strand not nicked becomes a circular DNA as it is. Here, when annealing is performed, most of the linear DNAs reassociate with each other or with single-stranded DNA on the circle. Remains without reunion. The remaining circular single-stranded DNA remains without reassociation. The remaining circular single-stranded DNA stochastically contains equimolar + and-strands and tries to reassociate, but cannot completely return to double strand due to steric hindrance. The remaining circular single-stranded DNA serves as a template, so that the sequencing reaction can proceed easily.

1) 5 μl of plasmid DNA in a microtube
2N NaOH 2 μl
2 mM EDTA 2 μl
D. W. 11 μl
Prepare 20 μl of Total.
2) Mix well and incubate at 37 ° C for 5 min.
3) Perform ethanol precipitation and dry.

DNA Sequence Determination
A method for examining a base sequence by synthesizing a complementary strand from a primer for a sequence located on both sides of a target DNA fragment using DNA polymerase. At the time of synthesis, nucleotide triphosphate (dNTP; dATP, dCTP, dGTP, dTTP) is put in the reaction solution as a raw material. At that time, fluorescently labeled dideoxynucleotides (ddNTP; ddATP, ddCTP) which are analogs thereof are used. , DdGTP, ddTTP). When this dideoxynucleotide is incorporated at a certain probability during DNA synthesis, synthesis stops there, and synthetic products of various lengths are produced. These fragments can be separated on a denaturing polyacrylamide gel that can also distinguish the difference in length of one base, and these can be subjected to capillary electrophoresis to meet different molecular weights. In this study, ABI PRISM 310 manufactured by Perkin Elmer was used as an automatic fluorescence sequencer.

1) Mix the PCR reaction solution for the sequencer and perform PCR under the following conditions. Change the primer as shown in the following table according to the analysis site.

Reaction composition Template DNA 0.4 μl
Dilution buffer 3.0 μl
Primer 4.0 μl
DMSO 1.0 μl
D. W. 9.6 μl
Big Dye (Ver1.1or3.1) (5 pmol / μl) 2.0 μl
Total 20 μl

PCR condition 98 ℃ ・ 5min
↓
Denatur 98 ℃ ・ 30sec
Annealing 50 ℃ ・ 15sec
Extension 60 ℃ ・ 4min * 25cycle repeat ↓
4 ℃

2) Add 2 μl of 3M CH ₃ COONa and 50 μl of 100% Ethanol, and let stand at room temperature for 15 min. All subsequent operations are performed with light shielding.
3) Centrifuge at 15000 rpm for 20 minutes or more.
4) Remove the supernatant with a pipette and add 200 μl of 70% Ethanol.
5) Centrifuge at 15000 rpm for 5 minutes.
6) Remove the supernatant with a pipette and dry under reduced pressure.
7) Add 20 μl of Hi-Diformamide.
8) After heating at 95 ° C. for 2 min, leave on ice for 5 min.
9) Transfer to a tube dedicated to the sequencer and perform gene sequence analysis with ABI PRISM 310.

[Transformation of actinomycetes and protein expression]
Preparation of actinomycete protoplasts Syringe (20 ml) stuffed with cotton, autoclaved and sterilized by dry heat (150 ° C, 2h)
・ The 250 ml centrifuge tube is sterilized with UV for 10 min. ・ 100 ml of 10.3% sucrose solution x number of cultures.

Basically, centrifugal operations and operations other than incubators are performed in a clean bench.
1) Put YEME medium 100ml + stainless steel coil into Sakaguchi flask and autoclave.
2) Shaking culture (pre-culture) for 2 to 3 days at 28 ° C. and 200 rpm in a test tube containing 3 ml of TSB medium with actinomycetes.
3) The pre-cultured bacteria were inoculated into a YEME medium and cultured with shaking at 28 ° C. and 200 rpm for 30 hours.
4) Transfer to a centrifuge tube, centrifuge at 8 ° C., 3000 rpm, 10 min, and discard the supernatant.
5) Add 50 ml of a 10.3% sucrose solution, suspend, centrifuge at 8 ° C., 3000 rpm, 10 min, and discard the supernatant (do this twice).
6) Add 15 ml of Lysozyme solution, suspend it, and leave it at 30 ° C. for 60 minutes or more while suspending it frequently.
7) Add 15 ml of P-buffer and suspend well.
8) Filter using a syringe stuffed with cotton and pre-claved.
9) Centrifuge at 8 ° C., 3000 rpm, 7 min, discard the supernatant, and add approximately 1500 μl of P-buffer. (Adjusted to some extent by the amount of apparent bacterial cells after filtration)
10) Dispense 50 μl each.
11) Store frozen in a deep freezer (-85 ° C).

Actinomycetes transformation (PEG method) and selection with antibiotics・ Prepare 15ml Corning for UV sterilization for the number of cultures ・ T-buffer 500μl x number of cultures ・ P-buffer 5.5ml x number of cultures ・Make an R2YE plate, dry it in a clean bench for 2-4 hours, and reduce the moisture by about 30%

Basically, centrifuge operations and operations other than incubators are performed in a clean bench. 1) Thaw a protoplast stock sample on ice.
2) Add 10 μl of plasmid solution and 200 μl of T-buffer and suspend.
3) Pour 100 μl each on R2YE plate and incubate at 30 ° C. for 1-2 days.
4) Add thiostrepton to 0.7% soft agarose (autoclaved by adding electrophoretic agarose LE classic type to DW) to a final concentration of 500 μg / ml, and overlay 3 ml on the plate. Incubate at 30 ° C. for 5 days.

Cultivation of transformants 1) Colonies that broke through the gel on the upper layer were picked with a platinum loop, and 3 ml of TSB medium (1.7% of pancreatic digest of casein, 0.3) containing thiostrepton (5 μg / ml) was added. Incubation is carried out for 2 to 3 days at 28 ° C. and 200 rpm in% of pacific digest of soybean meal, 0.25% of depth, 0.5% of sodium chloride, 0.25% dipotassium phosphate.
2) Add 3 ml of the seed culture to TSB medium (Sakaguchi flask) to which 100 ml of thiostrepton (5 μg / ml) is added, and culture at 28 ° C. and 200 rpm.
* Sampling is performed every 24 hours. For the sample, the bacterial cells and the culture solution are separated, and 1/100 vol of protease inhibitor is added to the culture solution and stored at 4 ° C.

[Protein manipulation and evaluation]
SDS-PAGE
SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is the simplest method for confirming the expression of a target protein. With SDS, the protein is denatured and at the same time negatively charged and migrates in a polyacrylamide gel, the protein moves from the cathode to the anode. The smaller the molecular weight, the longer the moving distance, and the larger the moving distance, the shorter the moving distance. Therefore, an approximate molecular weight can be estimated by comparing with a molecular weight marker. In addition, when the acrylamide in the gel is at a high concentration, a wide range of molecular weights can be covered, but the resolution of the polymer region decreases. When the concentration is low, the protein cannot be separated at a low molecular weight, but the resolution of the polymer region is improved.

Operation method Preparation of protein sample to be analyzed 1) Mix 2-mercaptoethanol (2-ME) and 2 × SDS Sample Buffer at 1: 9 (= 100 μl: 900 μl).
2) Mix the sample protein solution to be analyzed with the one made in 1) 1: 1 (= 15 μl: 15 μl).
3) After heat denaturation at 95 ° C for 3 minutes, cool with ice for 5 minutes or more.

Preparation of gel 1) The flat glass for electrophoresis, comb and packing are carefully wiped and assembled with 70% Ethanol.
2) A separating gel is prepared with the following composition. However, since the polymerization is started by mixing 10% APS and TEMED, 10% APS is added immediately before the start of the polymerization.

3) After stirring rapidly, the separated gel is poured into a gel molding plate to about half, and 70% Ethanol for sterilization is sprayed on it.
4) Next, a concentrated gel (Stacking Gel) is prepared with the following composition. Similar to Separating Gel, 10% APS is added just before the start of polymerization.

5) When the separated gel is solidified, remove the ethanol, and then pour the concentrated gel, and insert the comb so that no air bubbles enter on it.

Electrophoresis 1) Inject about 2 cm of 1 × SDS-Buffer into the electrophoresis tank, and set the gel plate in the electrophoresis tank, taking care not to allow air to enter the bottom of the gel plate.
2) Fill the inside of the gel plate with 1 × SDS-Buffer, and gently pull out the comb so as not to break the gel hole.
3) Using a micro syringe, apply 20 μl of Sample at a time to the gel hole.
4) Apply 5 μl of SDS-Marker (low molecular weight marker).
5) Set the power supply device to 1000 V, 20 mA / plate, and perform constant current electrophoresis.
6) Perform for about 2.5 h, and terminate the electrophoresis when the blue line reaches the bottom of Gel.

Detection of protein by CBB staining 1) After completion of SDS-PAGE, the gel is taken out, unnecessary portions are cut off, transferred to a container, and the CBB dye solution is added to the extent that the gel is completely immersed and stained for about 30 minutes.
2) Return the CBB dye solution to the storage container, put the destaining solution so that the gel soaks, and destain until an appropriate stained image is obtained.

Detection of protein by silver staining 1) After completion of SDS-PAGE, take out the gel, discard unnecessary parts, transfer to a container, add fixative I to the extent that the gel is completely immersed, and soak for 10 min or more (preferably about 1 h) .
* Fixing fluid I composition Methanol 200ml
Acetic acid 40ml
D. W. 160ml

2) Discard Fixing Solution I and immerse the gel in Fixing Solution II for 10 min or longer.
* Fixative II Composition Fixative I 47.5ml
Fixed stock solution 2.5ml

3) Discard Fixing Solution II and soak the gel for 10 min or longer with sensitizing solution.
* Sensitizer composition Sensitizing stock solution 2.5ml
Methanol 23.75ml
D. W. 23.75ml

4) Discard the sensitizing solution and immerse the gel in a large amount of distilled water for 10 min or longer.
5) Prepare the next staining solution during washing.
Staining solution A 2.5ml
Staining solution B 2.5ml
45ml distilled water

6) Discard the distilled water and immerse the gel accurately in the staining solution for 15 min.
7) Discard the staining solution in a dedicated waste container. Rinse once with distilled water.
8) Wash with distilled water. (Repeat 5 min 3 times).
9) Immerse the gel with developer. This starts the staining.
* Developer composition Stock solution 5ml
D. W. 47.5ml

10) Prepare a stop solution and add 2.5 ml to the container according to the degree of development. This stops the staining. Soak the gel for about 10 minutes.
11) Discard the solution in the container and wash with distilled water.

Western Blotting
Western blotting is a method in which after performing SDS-PAGE, the protein is transferred to a membrane and a specific protein is detected using an antibody. If an antibody against the protein of interest is available, it can be detected by this method.

  The antibody includes a polyclonal antibody and a monoclonal antibody. To prepare a polyclonal antibody, a rabbit or the like is immunized by injecting an antigen, and blood is collected after taking the place where the antibody is formed to obtain serum (antiserum). In Western blotting, it may be used as diluted as it is, or it may be used after purifying a specific antibody using an antigen column or the like.

Transfer of protein to membrane Prepared thing Semi-drive lotting device Power supply Plastic case Shaker Filter paper 6 sheets PVDF membrane TBS-T Buffer
1 x Transfer Buffer
Blocking Buffer

1) Immerse 8 pieces of filter paper with a size of about 7 × 10 cm in 1 × Transfer Buffer for about 5 minutes (× number of gels).
2) After immersing a PVDF membrane of about 7 × 10 cm in methanol for 5 min, transfer it to a container containing filter paper, and immerse it in 1 × Transfer Buffer for about 5 min. In the case of a nitrocellulose membrane, it is immersed in 1 × Transfer Buffer as it is without being immersed in methanol. * Do not touch the membrane with bare hands.
3) Transfer the SDS-PAGE gel to a container containing filter paper and membrane, and soak in 1 × Transfer Buffer for about 1 min.
4) Stack 4 sheets of filter paper, membrane, gel, and 4 sheets of filter paper from the anode side, taking care not to allow air bubbles to enter the device pre-wet with 1 × Transfer Buffer.
5) Set the power supply device at 150 V, 70 mA / gel (constant current), and transfer for 45 min.
6) Shake the membrane in a blocking buffer for 30 min or longer. SDS-Gel performs staining.
7) Discard the Blocking Buffer, put the TBS-T Buffer so that the membrane is completely immersed, shake for 5 minutes and wash about 3 times.

Preparations for reactions with antibodies Instruments Plastic case shaker

1) Discard the TBS-T Buffer, put the membrane in a nylon bag, and close the three sides with a policyr.
2) Put the cooled primary antibody in the bag and close the other side.
3) After stirring gently, react at 4 ° C. for 1 hr or more. (Normally overnight reaction)
4) Discard the primary antibody, put TBS-T Buffer so that the membrane is completely immersed, shake for 5 min and wash about 3 times.
5) Put the membrane in the nylon bag again and close the 3 sides with the policyr.
6) Put the cooled secondary antibody in the bag and close the other side.
7) After stirring lightly, react at 4 ° C. for 1 hour or more.
8) Discard the secondary antibody, put TBS-T Buffer until the membrane is completely immersed, shake for 5 min, and wash about 3 times.

Preparation for detection of antigen by alkaline phosphatase reaction Alkaline Phosphatase Buffer
NBT
BCIP

1) A coloring solution is prepared (5 ml for one membrane).
2) Put the membrane in a container and add the coloring solution.
3) When the band is visible, discard the coloring solution at an appropriate place and wash with H ₂ O.
4) Dry with a dryer.

[Purification of protein]
The most common methods for exchanging the buffer in the dialysis sample and desalting are dialysis and gel filtration. Dialysis uses a very thin (20-100 μm) regenerated cellulose porous membrane to exchange low molecules between the external and internal liquids by natural diffusion. For short-term dialysis, choose one that is as long as possible (having a large surface area).

Dialysis membrane Dialysis Membrane, Size20
Wako 546-00051 (small volume)
Seamless Cellulose Tubing
Sanko Pure Chemicals UC36-32-100 (large volume)

Buffer
20 mM Acetate Buffer (pH 5.6)
20 mM Phosphate Buffer (pH 7.0)

1) Since glycerol is contained as a preservative in commercially available dialysis membranes, put distilled water and dialysis membrane in a beaker, boil off for about 10 minutes, and wash with distilled water.
2) Tie the bottom of the dialysis membrane, add water, check for holes, and discard the water.
3) Put the sample with a pipette from the top (* 5ml or 50ml).
4) Tie off the top end with a few centimeters and hang it on the wall of the beaker.
5) Add 1 L of 4 ° C buffer and stir on a stirrer at 4 ° C.
6) After 4 hours or more, change the buffer of the external solution once and continue stirring for 4 hours or more.
* Change the buffer several times when using 50ml.
7) When dialysis is completed, transfer the sample from the top to Corning.

* Use 5 ml of culture supernatant for dialysis of PLD, and replace with 20 mM AcetateBuffer (pH 5.6).
* Globin and P450 are dialyzed using 50 ml of the culture supernatant and replaced with 20 mM Phosphate Buffer (pH 7.0).

Ion exchange chromatography principle ion exchange chromatography is one of the general purification methods may correspond to almost all soluble proteins. The total charge of the protein is negative at a pH above the isoelectric point and positive at a pH below the isoelectric point. On the other hand, carriers (gels) used for ion exchange chromatography include positively charged anion exchangers and negatively charged cation exchangers. Proteins with a negative total charge can bind to an anion exchanger and proteins with a positive total charge can bind to a cation exchanger. For elution of bound protein, a method of increasing the ionic strength (salt concentration) of the elution buffer is generally used. An increase in ionic strength (salt concentration) increases the counter ion binding to the ion exchanger. The counter ion binds to the ion exchanger instead of the protein, and is released and eluted in order from the weakly bound protein. As described above, the ion exchange chromatography is a chromatography performed by utilizing the electrostatic bond (ion bond) between the total charge of the protein and the dissociating group.

Equipment GRADENT PUMP BIO-RAD
ECONO UV MONITOR BIO-RAD
MODEL 2110 FRACTION COLLECTOR BIO-RAD

Globin, Purification of P450 Ion exchange column: HiTrapTMQ HP (1 ml) 17-1153-01 Amersham Biosciences AB
Starting buffer A: 20 mM Phosphate Buffer (pH 7.0)
Elution buffer B: 20 mM Phosphate Buffer (pH 7.0) +1.0 M NaCl

Operation method 1) Set the wavelength of the UV recorder to 280 nm.
2) Flow the starting buffer at a flow rate of 1 ml / min. The column is equilibrated by flowing 5 times the packed bed volume (15 min) of the starting buffer.
3) The salt is removed by dialysis and the sample (5-8) adjusted to the pH of the starting buffer is run twice at a flow rate of 1 ml / min and bound to the column.
4) Run the starting buffer at a flow rate of 1 ml / min for 10 minutes to wash the column.
5) The controller is set to create a NaCl concentration gradient (FIG. 4).
6) Start the fraction collector simultaneously with the start of elution.
7) The solution is fractionated with a 2 ml tube, and activity measurement and purity measurement (SDS-PAGE) are performed for each fraction.

Bradford method
The Bradford method is a method that utilizes a dye called Coomassie brilliant blue G-250 that binds to the basic and aromatic amino acid side chains of the protein, and as a result the absorption peak shifts from 465 nm to 595 nm. . Compared with other protein quantification methods, it is simple, rapid, and highly sensitive (0.02-0.1 mg / ml) and hardly affected by reducing agents and EDTA. Standard, buffer and target protein solution are measured, and protein concentration is calculated from the calibration curve. Bovine serum albumin (BSA) is used as a standard.

1) Add 50 μl of sample to D. W. Add 750 μl.
2) Add 200 μl of kid, vortex well, and leave at 37 ° C. for 15 min.
3) The absorbance at a wavelength of 595 nm is measured, and the protein concentration is quantified from a calibration curve.

<Example 1: Construction of foreign protein secretion expression vector with Streptomyces lividans 1326>
[Outline of gene recombination procedure used in vector construction]
1) Amplification of DNA fragment by PCR and 2nd PCR 2) Agarose electrophoresis of the entire amount of PCR product, and isolation of DNA insert using Gel purification kit. Alternatively, ethanol precipitation is performed to remove Buffer and the like.
3) Since the DNA insert has an arbitrary restriction enzyme site inserted into the primer, the vector is also treated with the corresponding restriction enzyme.
4) Isolate DNA fragment using Gel purification kit. Alternatively, the buffer and restriction enzyme are removed by ethanol precipitation.
* When using restriction enzymes with different buffers, repeat 3) and 4) to replace the buffers.
5) Perform Ligation.
6) Transform the Ligation product into E. coli Nova-Blue or DH5-a for gene cloning.
7) The formed colonies are inoculated into 3 ml of LB medium (+ Amp) and cultured overnight (Mini culture).
8) Purify the vector using alkaline I-II-III, isopropyl alcohol precipitation.
9) Sample is RNase-processed.
10) Perform agarose electrophoresis and select a vector that seems to have an insert.
11) Confirm whether the target insert was incorporated by restriction enzyme treatment or PCR Check.
12) Polyethylene glycol precipitates the sample in which the insert is confirmed.
13) Perform Sequence and confirm the base sequence. Sample storage is performed at -20 ° C.

[Overall Construction Procedure] (FIGS. 1 and 5)
i) A primer with an arbitrary restriction enzyme site was designed and actinomycetes Stv. A translation stop region (Terminator region) downstream of the cinnamonium chromosome-derived PLD gene is amplified by PCR and incorporated into pUC18 having a multiple cloning site. (PUC18 + term)

ii) The sequence coding for Ala and Ser at the end of the signal region was changed to the NheI site, a primer with an arbitrary restriction enzyme site was designed, and actinomycetes Stv. The promoter region and signal region upstream of the cinnamoneum chromosome-derived PLD gene are amplified by the PCR method and incorporated into pUC18 + term. (PUC18 + promoter + terminator)

iii) A plasmid having the gene of the target protein and a PCR fragment as a template, a primer having an Nhe site at the N-terminus of the gene and an arbitrary restriction enzyme site at the C-terminus are designed, amplified by PCR, and pUC18 + promoter + terminator The pld gene is integrated into other foreign protein genes.

vi) The entire length of the promoter region, signal region, foreign protein gene, and terminator region is excised from the vector and incorporated into the pUC702 vector, which is an E. coli / actinomycetes shuttle vector. (PUC702-pro-XXX)

A method for preparing a DNA fragment to be amplified is shown below.
Production of Promoter and Signal Regions The Promoter and Signal regions are amplified by PCR under the following conditions.
・ Use Template pUC18-pro-PLD
・ Primer
S-HindIII-promoter (SEQ ID NO: 7)
5'- CCCAAGCTTACGTCATGGCGGGTCTCTCTCGTC-3 '
As-BamHI-NheI-signal (SEQ ID NO: 8)
5 '-CCCGGATCCGCTAGCGAAGGCCGGAGCCGCGGGCAG -3'

-Reaction composition Template 0.25 μl
Primer (100 pmol / μl) 0.75 μl each
dNTP (2.5mMeach) 2.0μl
× 5 Buffer 5.0 μl
D. W. 16.0 μl
Poly (Takara Prime STAR HS)
0.25 μl
Total 25.0 μl

・ Reaction condition (Hot Start)
98 ° C for 1 min
↓ Add polymerase 98 ℃ 1min
↓
Denatur 98 ℃ ・ 30sec
Extension 72 ℃ ・ 2min * 25cycle repeat ↓
72 ° C 3min
↓
4 ℃

Various protein genes are amplified by PCR under the following conditions.
Table 18 shows the template and primer used, the extension time of PCR, and the restriction enzyme depending on the protein gene. * The remaining conditions are the same.

-Reaction composition Template 0.25 μl
Primer (100 pmol / μl) 0.75 μl each
dNTP (2.5 mMeach) 2.0 μl
× 5Buffer 5.0μl
D. W. 16.0 μl
Poly (Takara Prime STAR HS)
0.25 μl
Total 25.0 μl

・ Reaction condition (Hot Start)
98 ° C for 1 min
↓ Add polymerase 98 ℃ 1min
↓
Denatur 98 ℃ ・ 30sec
Extension 72 ° C / Xmin * 25 cycles repeated ↓
72 ℃ ・ X + 0.5min
↓
4 ℃

  * The vector of pUC18 + promoter + terminator was prepared by replacing the amino acid of the sequence encoding Ala and Ser at the C-terminal of the signal region with the restriction enzyme site NheI. When the gene is translated into protein and secreted, amino acids are cut behind this Ser. Therefore, it is possible to secrete and express only the target protein by adding a NheI site to the N-terminus of the protein gene to be expressed, amplifying the DNA fragment, and inserting it (FIG. 6). In FIG. 6, the upper base sequence is SEQ ID NO: 48, the upper amino acid sequence is SEQ ID NO: 49, the sequence on the left side of the interruption is SEQ ID NO: 50, the sequence on the right side of the interruption is SEQ ID NO: 51, SEQ ID NO: 52 is the left side of the omitted part of the lower base sequence, SEQ ID NO: 53 is the right side of the omitted part of the lower base sequence, SEQ ID NO: 54 is the left side of the omitted part of the lower amino acid sequence. The right side of the omitted part of the amino acid sequence is represented by SEQ ID NO: 55.

<Example 2: Expression of foreign protein>
In order to express foreign proteins in an expression system of actinomycetes capable of secreting and expressing PLD in large quantities, a vector in which the PLD gene of pUC702-pro-pld was replaced with a foreign protein gene was constructed (FIG. 5). lividans was transformed and foreign protein expression was attempted with actinomycetes.

[ About GFP and BFP ]
Similarly to the case where PLD was expressed, the transformant of the vector replaced with the above protein gene was also cultured in TSB medium at 28 ° C. for 72 h, and the culture supernatant and the protein in the bacterial cell were analyzed.

Results and Discussion First, proteins were separated by SDS-PAGE, and protein expression was confirmed. However, no band dominant in the size of the target protein was observed, and it was found that the mass secretion expression (50-100 mg / l) was not performed as in the case of PLD expression.

Therefore, Western blotting was performed using a GFP antibody (FIG. 7). BFP was not detected inside or outside the bacteria. GFP was detected inside and outside the fungus. However, a band similar to the size of GFP and a very low band were detected inside and outside the cells. The low-size band is considered to be a result of GFP degradation. It can be seen that the band density decreases in the order of a low band inside the cell, a GFP band inside the cell, a GFP band outside the cell, and a low band outside the cell.

  From these, although GFP is expressed, most of them are considered to be decomposed in the microbial cells and secreted in a very small amount. Although the discussion from the data is above, various mutants of GFP have been made, and recombination has been studied in various organisms (Miyawaki Atsushi, GFP and Bioimaging Yodosha (2002)). reference). When the knowledge is referred to, it can be mentioned that it becomes an insoluble fraction in the microbial cells with respect to non-expression.

  When GFP is expressed in a microorganism, it is said that if the protein synthesis rate is too high, GFP is not folded but accumulated as an inclusion body in the cell. The actinomycetes expression system we used is applicable. In addition, GFP orientation is unsuitable depending on the species, and mutants such as wtGFP, GFPuv, and GFPmut3.1 are suitable for microorganisms. In animal and plant cells, the nucleotide sequence is optimized in accordance with human codon usage. The body EGFP is suitable. Since all the GFP used this time is EGFP, this is also considered to be one of the factors.

[About globin; A1, A2, B1, B2, P450; CYP102A3, CYP106A2]
Similarly to the case where PLD was expressed, the transformant of the vector replaced with the above protein gene was also cultured in TSB medium at 28 ° C. for 72 h, and the culture supernatant and the protein in the bacterial cell were analyzed.

Results and Discussion First, proteins were separated by SDS-PAGE, and protein expression was confirmed. However, no band dominant in the size of the target protein was observed, and it was found that the mass secretion expression (50-100 mg / l) was not performed as in the case of PLD expression.

  Therefore, Western blotting was performed using a globin antibody (FIG. 8). * Since there is no P450 antibody, Western blotting is not possible. A1 was not detected. A2, B1, and B2 were detected in the target bands. Thus, there is little report of recombinant protein expression with a heme-binding region (Tong-Fian Shen, Chien Ho; Production of unmodified human adglobin in Escherichia coli [Proc. Nat. USA Vol.90, 8108-8112.1993]) a protein called globin could be expressed in an actinomycetes expression system. From this result, it was possible to show the possibility of expression of a complex three-dimensional structural protein having a heme-binding region such as NO synthase, chloroperoxidase, and CooA (Tsuneo Omura, P450 Molecular Biology Kodansha (2004)).

Examination of culture conditions When globin was expressed in TSB medium, the expression level was confirmed, but the expression level was very small. Therefore, hemin necessary for globin to form a three-dimensional structure was found in actinomycetes. It is thought that it is not fully synthesized. In the expression system of Escherichia coli, hemin (Non-patent Document 2) and 5-amino levulinic acid hydrochloride (5-ALA) (Ingela Jansson, John B. Schenkman; Enhanced expression of Y1) Since there was a report that the expression was induced by adding Escherichia coli [Toxiology 144, 211-219.2000]), the culture conditions were examined. Since there are many restrictions on addition of hemin, first, 5-ALA addition was tried. During the main culture, 5-ALA was added to the TSB medium so as to have a concentration of 2.5 mM, and the culture was similarly performed at 28 ° C. for 72 hours, and the culture supernatant and proteins in the cells were analyzed.

Results and Discussion After culturing for 72 hours, when the culture supernatant and bacteria were separated by centrifugation, all culture supernatants of globin and P450 turned red. Then, as a result of analyzing similarly by SDS-PAGE and Western blotting, expression was not confirmed by SDS-PAGE. However, A2, B1, and B2 were detected by Western blotting (* 5, not shown because it is almost the same as no ALA added). Since the medium was red, it was assumed that the medium was expressed, and purification was performed in order to analyze the concentration of expressed globin and P450, CD-spectrum, and an absorptiometer.

Purification of globin 50 ml of culture supernatant was subjected to buffer exchange and desalting by dialysis, and purified by anion exchange chromatography. From the liquid color of each fraction, a red-colored fraction was found and confirmed to be purified by SDS-PAGE.

Results and Discussion As a result of purification, a pink liquid was purified into fractions 21 to 25 for all six types. Proteins of fractions 21-25 were analyzed by SDS-PAGE. Proteins were confirmed at the height of the band that was neither globin nor P450 for all six types (not shown). Therefore, for confirmation, S. cerevisiae not transformed is used. When lividans was cultured in the same medium added with 5-ALA, the medium turned red. That is, by adding 5-ALA to the culture medium, It is thought that induction of secreted heme-binding protein in which lividans is on chromosomal DNA occurred. Therefore, it was shown that even when 5-ALA was added to the medium, the expression of globin, P450 was not changed.

<Conclusion>
From the results of the above examples, the expression of foreign proteins was examined in an actinomycetes expression system using a vector in which the PLD gene of pUC702-pro-pld constructed for the first time in the present invention was replaced with a foreign protein gene. It was revealed that secretion expression of GFP, globin; A2, B1, and B2 is possible.

  Since these GFP, globin; A2, B1, and B2 are foreign proteins derived from organisms other than actinomycetes, the PUC702-pro-pld PLD gene constructed for the first time in the present invention was replaced with a foreign protein gene. The vector is considered to be a vector that allows suitable expression and secretion in actinomycetes even if it is a foreign protein derived from organisms other than actinomycetes such as mammals and Bacillus subtilis.

  In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

For example, in the above embodiment, copper is used as the wiring material, but an alloy containing copper and silver may be used. in this case,···
In addition, as a low dielectric constant film ...

It is the schematic diagram which showed the scheme which inserts a green fluorescent protein (GFP) gene in the vector (pUC18 + promoter + term) used in the Example. It is a figure for demonstrating the effect | action of the promoter area | region in the secretion of PLD in actinomycetes using pUC702-promoter-PLD. It is a figure for demonstrating the secretion mechanism of the protein by Sec-pathway and SRP-pathway in actinomycetes. It is a schematic diagram for demonstrating the salt concentration gradient of the elution buffer in an ion exchange chromatography. It is the schematic diagram which showed the scheme for demonstrating construction | assembly of the expression vector which secretes a foreign protein by the actinomycete Streptomyces lividans 1326 used in the Example. A method for amplifying a DNA fragment by adding an NheI site to the N-terminus of a protein gene desired to be expressed in the vector described in FIG. 5 and inserting it into a secreted protein. It is a schematic diagram which shows an arrangement | sequence. It is the photograph which showed the result of SDS-PAGE at the time of expressing GFP, BFP, and GST-GFP with actinomycetes using the vector demonstrated in FIG. It is the photograph which showed the result of SDS-PAGE at the time of expressing globin; A1, A2, B1, B2, P450; CYP102A3, CYP106A2 by actinomycetes using the vector demonstrated in FIG. Hopwood, D.H. A. Group and Cohen, S .; N. It is the restriction map of pIJ101 developed by the group.

Claims (14)

A vector for expressing a foreign protein derived from an organism other than actinomycetes in actinomycetes,
A promoter sequence of a phospholipase D gene derived from Streptobacillus cinnamonium;
A signal sequence of the phospholipase D gene derived from Streptobacillus cinnamonium that is operably linked downstream of the promoter;
A foreign base sequence encoding a foreign protein, operably linked downstream of the signal sequence;
A terminator sequence of a phospholipase D gene derived from Streptobacillus cinnamonium linked downstream of the foreign protein;
A vector comprising: The vector of claim 1,
The promoter sequence is a vector comprising a base sequence in which one or several bases are deleted, substituted or added in the promoter sequence described in SEQ ID NO: 1. The vector according to claim 1 or 2,
The terminator sequence is a vector comprising a base sequence obtained by deleting, substituting, or adding one or several bases in the terminator sequence shown in SEQ ID NO: 2. The vector according to any one of claims 1 to 3,
The signal sequence consists of a base sequence encoding an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence of the signal peptide encoded by the signal sequence shown in SEQ ID NO: 3. vector. The vector according to any one of claims 1 to 4,
The vector, wherein the foreign base sequence is a base sequence encoding a foreign protein derived from a mammal or Bacillus subtilis. The vector of claim 5,
The vector, wherein the foreign base sequence is a base sequence encoding one or more foreign proteins selected from the group consisting of GFP and globin; A2, B1, and B2. The vector according to any one of claims 1 to 6,
A vector that is a plasmid. The vector according to any one of claims 1 to 7,
A vector further comprising an origin of replication sequence that allows the vector to replicate in the actinomycetes. The vector according to any one of claims 1 to 8,
It further comprises an origin of replication sequence that allows the vector to replicate in E. coli. vector. The vector according to any one of claims 1 to 9,
A vector further comprising an antibiotic resistance gene. A vector for expressing a foreign protein in actinomycetes by incorporating a foreign protein derived from an organism other than actinomycetes,
A promoter sequence of a phospholipase D gene derived from Streptobacillus cinnamonium;
A signal sequence of the phospholipase D gene derived from Streptobacillus cinnamonium that is operably linked downstream of the promoter;
A cloning site for inserting a foreign base sequence encoding the foreign protein provided downstream of the signal sequence;
A terminator sequence of a phospholipase D gene derived from Streptobacillus cinnamonium linked downstream of the cloning site;
A vector comprising: Recombinant actinomycetes for expressing foreign proteins derived from organisms other than actinomycetes,
Recombinant actinomycetes in which the vector according to any one of claims 1 to 10 is incorporated. Recombinant actinomycetes according to claim 12,
Recombinant actinomycetes, which is Streptomyces lividans. A method for producing foreign proteins derived from organisms other than actinomycetes in actinomycetes,
Culturing the recombinant actinomycetes according to claim 12 or 13 in a medium;
Obtaining the foreign protein from the cells or medium of the recombinant actinomycetes,
Including a method.