KR101431084B1 - Method for producing active inclusion body using PoxB gene - Google Patents

Abstract

The present invention provides a fusion protein comprising a fusion gene comprising a PoxB gene and a gene encoding a target protein, a fusion protein encoded by the fusion gene, an expression vector comprising the fusion gene, A transformant in which an expression vector is transformed into a host cell, and a method of producing a target protein comprising the step of producing an active inclusion body of a target protein using the PoxB gene. Since the active inclusion body produced according to the present invention shows the activity of the protein without a separate process, it is not necessary to produce the enzyme, separate purification and enzyme immobilization step in the industrial process requiring the enzyme reaction, Can be reduced, and the disadvantage that the enzyme activity is rapidly reduced during the immobilization process can be compensated, and the purified protein can be easily separated.

Description

A method for producing an active inclusion body using a PoxB gene (PoxB gene)

The present invention relates to an active inclusion body production method of a target protein using PoxB gene. More specifically, the present invention relates to a fusion protein comprising a fusion gene in which a PoxB gene and a gene encoding a desired protein are bound, a fusion protein encoded by the fusion gene, and a fusion protein comprising the fusion gene, An expression vector, a transformant obtained by transforming the expression vector into a host cell, and an active encasing body of the target protein using the PoxB gene.

Microorganisms are mainly used industrially for mass production of useful substances such as chemical substances and enzymes, and food fermentation industries. Methods have been used to classically mutate host bacteria through treatment of mutagen inducers, ultraviolet light, or irradiation to produce large quantities of useful enzymes or proteins, but these methods often result in the growth rate of host bacteria Degrade, and it is often difficult to optimize medium composition and culture conditions. In addition, in the case of mutant strains, strain degeneration occurs and a host microorganism for producing each target protein must be separately developed, which is a classic method that requires a lot of time and effort. Therefore, recently, a method of increasing the productivity of a desired protein or material by making a recombinant microorganism using genetic engineering technology has been attempted, and a method of inducing overexpression of a target protein using a strong promoter has become a basic. However, excessive overexpression of the target protein forms an inclusion body (IB) to produce an inactivated protein, and thus it is necessary to control the expression amount of the target protein to an optimal state.

When the target protein is overexpressed in the microorganism as described above, a crystal called an inclusion body is produced. In such an inclusion body, it is general that the expressed protein loses activity of the protein as a result of erroneous folding. The formation of encapsulated bodies is a major obstacle to the mass expression of proteins, and it is difficult to dissolve the formed encapsulated body and turn it into an active protein again, which consumes additional costs.

Thus, several strategies have been attempted to avoid the production of such inclusion bodies during protein expression. In the case of expression of a fusion protein containing? -Galactosidase in E. coli, a method of regulating these conditions has been developed since the temperature and pH of the culture medium have a great effect on the formation of inclusion bodies, Studies have also shown that reducing the formation of inclusion bodies of fusion proteins in a high-cell density fed-batch mode. In addition, fusion tags technology has been developed to increase the solubility of expressed proteins in the cytoplasm and the purification efficiency of expressed proteins as an alternative approach to inhibition of inclusion body formation (de Groot, NS and S. Ventura, 2006. FEBS Lett. 580: 6471-6476). Particularly, as a method for preventing protein aggregation, molecular chaperons related to protein refolding and protein secretion have been examined from the viewpoint of coexpression of molecular chaperone and aggregation-tendency protein (Bukau, B., J. et al. et al. 2006. Cell 125: 443-451).

However, although many studies have been made on the production of such inclusion bodies, the precise mechanism of inclusion body formation is not yet known, and no general association has been found. The formation of the inclusion body varies depending on the host-vector system, the characteristics of the protein, the culture conditions and the expression conditions, and thus can be known only through experiments in a desired system. Studies have been conducted on the case where an encapsulated body forms an active inclusion body without losing its activity.

The formation of such an active inclusion body has industrial significance. For example, in a chemical reaction using enzymes, it is necessary to immobilize a specific substance so that it can be easily separated for repeated reuse of the enzyme. In the case of the active inclusion body, the enzyme having already active forms crystals, not. Therefore, since the enzyme concentration, pure water separation, and chemical immobilization steps necessary for enzyme immobilization are not required, the process cost is reduced, and the disadvantage that the enzyme activity is rapidly reduced during the enzyme immobilization process can be prevented in advance. In addition, it can be used for the overexpression of a protein having toxicity of a cell, and the purified protein can be easily separated. However, occlusion bodies are often inactive in many cases and require additional processing accordingly.

Recently, the capsid protein VP1 of the foot-and-mouth disease virus, the human beta-amyloid peptide Ab42 (F19D), the maltose-binding protein mutant MalE31, Several cases have been reported in which a fusion protein with a suitable protein, such as a cellulose-binding domain CBDclos, forms an encapsulated body with activity.

Under these circumstances, the present inventor has made intensive efforts to develop a protein that can be used without additional processing by forming an encapsulated body having an activity. As a result, when the PoxB gene is bound to a gene encoding a target protein, Clms Page number 16 > body, and completed the present invention.

It is an object of the present invention to provide a fusion gene in which a PoxB gene and a gene encoding a target protein are bound to produce an active inclusion body of a target protein.

It is another object of the present invention to provide a fusion protein encoded by the fusion gene.

It is another object of the present invention to provide an expression vector comprising the fusion gene.

It is still another object of the present invention to provide a transformant in which the expression vector is transformed into a host cell.

It is another object of the present invention to provide a method for producing a target protein comprising the step of producing an active inclusion body of a target protein using the poxB gene.

In order to accomplish the above objects, the present invention provides a fusion protein comprising a PoxB gene and a gene encoding a target protein, the fusion gene for producing an inclusion body of an active protein of interest, Lt; / RTI > protein.

In the present invention, the term "PoxB gene" means a gene encoding pyruvate oxidase belonging to an oxidoreductase, and the PoxB enzyme produces acetyl phosphate using pyruvate and phosphate as substrates. For the purpose of the present invention, the PoxB gene is used to induce production of an active inclusion body when a target protein is overexpressed in a microorganism such as Escherichia coli.

The PoxB gene may be any gene encoding an enzyme having pyruvate oxidase activity. Preferably, the PoxB gene may be derived from Paenibacillus polymyxa. More preferably, the PoxB gene has a nucleotide sequence of SEQ ID NO: 1 . In addition, the protein encoded by the PoxB gene may preferably have the amino acid sequence of SEQ ID NO: 2.

In one embodiment of the present invention, when the PoxB protein derived from Penny Bacillus polymyx was overexpressed in E. coli, it was confirmed that the encapsulated body forms an inclusion body and that the formed encapsulated body has an activity One). Accordingly, it was confirmed that when the PoxB gene is bound to a gene encoding a target protein, the formation of an active inclusion body of a target protein can be induced (FIGS. 2 and 3).

In the present invention, the term "target protein" means a target protein to be expressed, and for the purpose of the present invention, a protein which is a target for producing an active inclusion body in order to maintain its activity. The gene coding for the target protein binds to the PoxB gene of the present invention to form a fusion gene, thereby forming a clustering body of the target protein, thereby exhibiting protein activity without further immobilization. In one embodiment of the present invention, GFP (green fluorescent protein) and amylase are used as the target protein, and when the GFP is used as a target protein, the fusion gene of the present invention preferably has a nucleotide sequence of SEQ ID NO: 5 When the amylase is used as a target protein, the fusion gene of the present invention may preferably have the nucleotide sequence of SEQ ID NO: 9.

In the present invention, the term "fusion gene" means a gene in which different genes are linked by a method such as gene recombination, and is generally used to produce a fusion protein having higher utility than the conventional one. For the purpose of the present invention, the fusion gene means a PoxB gene and a gene encoding a target protein bound thereto, thereby producing an active inclusion body of the target protein. The method of fusion may be any fusion method commonly used in the art, for example, a method of genetic recombination. The term "fusion protein" also refers to a protein in which some or all of two or more heterologous proteins are combined. The fusion protein is a fusion protein encoded by the fusion gene for the purpose of the present invention. In the case of using GFP as the target protein, the fusion protein may have the amino acid sequence of SEQ ID NO: 6, and when amylase is used as the target protein, May have an amino acid sequence.

In the present invention, the term "inclusion body" refers to a structure having a nucleus or protoplast staining characteristic. When the recombinant protein is produced in E. coli or yeast, it does not secrete into the medium, It is also called an inclusion body or an inner body. Overexpression of a protein in a microorganism results in the formation of a crystal called an inclusion body. The inclusion body is a result of an incorrect folding of the expressed protein, which generally results in loss of activity of the protein. Therefore, since the encapsulation body is required to be an active protein again, an active protein can be produced more efficiently by producing an active inclusion body without losing the activity of the protein without such a process. Thus, in the present invention, the term "active inclusion body" means an inclusion body maintained without loss of protein activity. In the present invention, it was confirmed that poxB has poxB activity when forming an inclusion body, and when it is bound to a target protein, it is confirmed that a closure body of the target protein can be produced.

In one embodiment of the present invention, a PoxB _pp -gfp fusion gene (SEQ ID NO: 5) and a PoxB _pp- amyE fusion gene (SEQ ID NO: 6) were prepared to confirm formation of an inclusion body of the target protein and its activity . First, a plasmid containing the PoxB _pp- gfp fusion gene was prepared, introduced into E. coli, cultured, and observed under optical and fluorescence microscope. As a result, the formation of an active inclusion body and observation of fluorescence revealed that an active inclusion body was produced (Fig. 1). In addition, a plasmid containing the PoxB _pp- amyE fusion gene was prepared, introduced into Escherichia coli, and cultured. The resultant was observed under an optical microscope. As a result, it was confirmed that an encapsulated body was produced, and the amylase activity was further measured. As a result, PoxB-AmeE In the fusion protein group, it was confirmed that the amylase enzyme activity was high in the precipitate containing the inclusion body (FIG. 2), thereby confirming that the fusion of the PoxB gene induces formation of a clustering body of the target protein.

In another embodiment, the present invention provides an expression vector comprising the fusion gene, and a transformant in which the expression vector is transformed into a host cell. The fusion gene is a fusion gene in which a PoxB gene and a gene encoding a target protein are combined to produce an active inclusion body of a target protein, as described above.

The term "vector" in the present invention means a nucleic acid molecule capable of carrying other nucleic acid to which it is linked. One type of vector, "plasmid ", is a circular double- stranded DNA Loop.

As used herein, the term "expression vector" means a recombinant vector capable of expressing a target protein in a suitable host cell, and which expresses a gene encoding a target protein operably linked. Such expression vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors such as adenovirus vectors and retroviral vectors, and may be plasmid vectors, preferably in the use of recombinant DNA technology .

In the present invention, the term "transformation" refers to a phenomenon in which DNA is introduced as a host and DNA can be cloned as a factor of chromosome or by integration of chromosome integration, thereby introducing an external DNA into cells to cause an artificial genetic change it means. The host cell of the present invention may be any host cell known in the art without limitation. In one embodiment of the present invention, Escherichia coli was used as a host cell.

Any transformation method of the present invention can be used, and can be easily carried out according to a conventional method in the art. Generally, the transformation methods include the CaCl ₂ precipitation method, the Hanahan method which uses a reducing material called DMSO (dimethyl sulfoxide) for the CaCl ₂ method, the electroporation method, the calcium phosphate precipitation method, the protoplasm fusion method, Agrobacterium-mediated transformation, transformation with PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods. Therefore, in the present invention, a transformant can be obtained by introducing an expression vector containing the fusion gene of the present invention into a host cell by using the above transformation method without limitation.

In another aspect, the present invention provides a method for producing a fusion protein comprising the steps of: (a) preparing a fusion gene by binding a PoxB gene to a gene encoding a target protein to produce an active inclusion body; (b) preparing an expression vector comprising the fusion gene; (c) preparing a transformant transformed with the expression vector prepared in the host cell; (d) culturing the transformant to produce an active inclusion body of the target protein.

The method of the present invention may further comprise (e) cleaving the target protein in the produced active inclusion body.

The present invention provides a method for producing a target protein by producing an active inclusion body of a target protein using a PoxB gene capable of inducing formation of an active inclusion body. The fusion protein of the present invention, the expression vector , Transformants, and active inclusion bodies are as described above.

The encapsulated body of the target protein produced by culturing the transformant is active and can be used as a protein having no activity such as enzyme immobilization. The target protein includes, without limitation, proteins that are intended to produce an active inclusion body to maintain its activity, such as GFP and amylase.

Since the active inclusion body produced according to the present invention shows the activity of the protein without a separate process, it is not necessary to produce the enzyme, separate purification and enzyme immobilization step in the industrial process requiring the enzyme reaction, Can be reduced, and the disadvantage that the enzyme activity is rapidly reduced during the immobilization process can be compensated, and the purified protein can be easily separated.

Figure 1 shows the formation of active inclusion bodies of PoxB. FIG. 1 (A) is a micrograph showing the formation of an encapsulated body by overexpression of poxB (poxB _pp ) by Penny Bacillus Polychyx in Escherichia coli. B represents a wild type Escherichia coli (BW25113), poxB deletion mutant (poxB) (PUC-pppox) -sol), and a precipitate (poxB (pUC-pppox) -insol).
2 is an optical microscope (A) and a fluorescence microscope (B) photograph of E. coli into which the poxB-gfp fusion gene is introduced.
FIG. 3 is a photograph (A) of the E. coli into which the poxB-amyE fusion gene is introduced and a graph (B) showing the activity of the amylase enzyme activity. sol: supernatant; insol: precipitate, amy: amyE protein; pox-amy: poxB-amyE fusion protein; -xylose: absence of xylose, + xylose: xylose present.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  One: PoxB of Inclusion body ( inclusion body , IB ) Formation Confirmation

In order to overexpress poxB protein derived from Paenibacillus polymyxa (poxB _pp ) in Escherichia coli, the poxB gene (SEQ ID NO: 1) was obtained from the genome of Penny Bacillus polymyxer strain E681 (J. Bacteriol. 2010. 192: 6103-6104) ). The poxB gene was obtained by PCR using the poxB-hind primer (SEQ ID NO: 2) and the poxB-bam primer (SEQ ID NO: 3), and the obtained PCR product was digested with BamHI and HindIII restriction enzymes and then ligated to the same site of the plasmid pUC19 To prepare pUC-pox. The prepared plasmid pUC-pox was introduced into Escherichia coli BW25113 (Proc. Natl. Ac. Sci. USA 97: 6640-6645) and poxB deletion mutant JW0855 (Mol. BW25113 containing pUC-pox was cultured in LB solid medium for 2 days and then observed with a microscope. As a result, it was confirmed that poxB was overexpressed in E. coli to form an inclusion body (IB) (FIG. 1A).

Next, BW25113 and JW0855 containing pUC-pox were cultured in LB solid medium for 2 days and then cells were collected to confirm that the encapsulated body formed above had poxB activity. Cells were then disrupted with sonication and centrifuged to separate the supernatant and precipitate. 1982. 151: 1279-1289 (Chang, Cronan). At this time, the enzyme activity of poxB is lowered at A450 nm.

The activity of poxB was measured in the supernatant (sol) and the precipitate (insol) obtained by centrifuging the BW25113 wild type Escherichia coli strain, poxB deletion mutant JW0855, poxB deficient mutant strains by introducing poxB _pp , PoxB activity was observed not only in the supernatant but also in the precipitate (Fig. 1B). This means that the inclusion body of poxB formed in the above is an inclusion body having activity.

Example  2: GFP Active Inclusion body  making

Was PoxB _pp is confirmed, _pp PoxB and gfp a PoxB _pp fusion genes -gfp fusion gene (SEQ ID NO: 5) for making the activity of the inclusion bodies by induction in order to ensure that the active inclusion body formation of other proteins .

First, PoxB _pp was obtained by PCR using the poxBF-apa primer (SEQ ID NO: 7) and the poxBR-xho primer (SEQ ID NO: 8) from the genome of the Penny Bacillus policeman E681 strain. The obtained PCR products were ApaI and XhoI After digestion with restriction enzymes, the plasmid pSG1164 (gene 1999. 227: 101-109) containing the gfp gene was cloned into the same site to prepare pSG-pox. The prepared plasmid pSG-pox was introduced into Escherichia coli BW25113 and cultured in LB solid medium for 2 days, followed by optical and fluorescence microscopy.

As a result, formation of an encapsulated body under an optical microscope was observed (A in FIG. 2), which confirmed fluorescence under a fluorescence microscope (FIG. 2B). These results confirmed that the PoxB-Gfp inclusion body is an active inclusion body. When the poxB gene is fused to the gene encoding the target protein, it is possible to induce the formation of the active inclusion body of the target protein .

Example  3: Amylase  activation Inclusion body  making

Was PoxB _pp is confirmed, _pp PoxB and amyE a PoxB _pp fusion genes -amyE fusion gene (SEQ ID NO: 9) for making the activity of the inclusion bodies by induction in order to ensure that the active inclusion body formation of other enzyme .

First, the amyE gene was obtained by PCR using the amyF-xho primer (SEQ ID NO: 11) and the amyR-spe primer (SEQ ID NO: 12) from the genome of Bacillus subtilis (Nature 1997. 390: 249-256). Next, PoxB _pp was obtained by PCR using the poxBF-apa primer (SEQ ID NO: 7) and poxBR-xho primer (SEQ ID NO: 8) from the genome of the Penny Bacillus polymyxer strain E681 and the amyE gene and PoxB _pp XhoI. Then, the plasmid pSG1164 was digested with ApaI and SpeI restriction enzymes and cloned into the same site of plasmid pSG1164 to prepare pSG-poxamy. The amyE gene used as a control was obtained by digesting the PCR product obtained using the amyF-xho primer and the amyR-spe primer with XhoI and SpeI, and then cloning into the same site of the plasmid pSG1164 to prepare pSG-amy. The prepared plasmids were introduced into Escherichia coli BW25113, cultured in LB solid medium for 2 days, and then observed with an optical microscope. As a result, it was observed that PoxB-AmyE forms an inclusion body (Fig. 3A).

Next, in order to confirm that the encapsulated body formed above had activity, amylase enzyme activity was measured. The cultured cells were collected, and the cells were disrupted by sonication and centrifuged to separate the supernatant and the precipitate. The amylase activities of these supernatants and precipitates were measured by the DNS method (Harwood & Cutting, 1990. Molecular biological methods for Bacillus, John Wiley & Sons Ltd. p380-381). amyE gene and the poxB-amyE fusion gene were respectively measured in the group to which the protein expression inducing substance xylose or no xylose was added.

As a result, amyE showed the most activity in the supernatant and little activity in the precipitate, whereas the PoxB-AmyE fusion protein had higher amylase activity in the precipitate containing the inclusion body. In addition, similar pattern results were observed regardless of the presence or absence of xylose (Fig. 3B). In this way, it was confirmed that the inclusion body formed by PoxB-AmyE is an active inclusion body. When the poxB gene is fused to the gene encoding the target enzyme, it is possible to induce the formation of the active inclusion body of the target enzyme It means that you can do it.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for producing active inclusion body using PoxB gene <130> PA110883KR <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 1725 <212> DNA <213> Paenibacillus polymyxa <400> 1 atgaagacaa tcgcagatac tattgtacaa gttttggtca atgcaggggt caagcggatt 60 tatggcattg ttggagactc tctaaataat atggttgatt ccattcgcag caatggtcaa 120 atcgaatgga ttcatgtaag gcacgaagaa gtggctgcct ttgcagccgg agcggatgct 180 gaccttagtg gcagcattgc tgtatgtgct ggaagtagtg gtcccggaaa tttgcatctg 240 attaatggtt tatatgattg ccaccgcaat cgggtgcctg tactggctat tgccgctcat 300 attccaagcg acgaaatcgg aagtgaatat tttcaagcga cgcatcctga gcatcttttc 360 ggagaatgca gtcacttttg tgaggttatt acgacaccgc gtcaaattcc cagaacggtg 420 accatggcca ttcaacaggc aatttcacgt tcaggtgtct ccgtcattgt tcttcccggt 480 gatgtagcag ctttggaggc ggaaaaggta cccatccctg aacatgtcta ccatcccact 540 gcacctgtag tgcatccgtc agcttccgaa atttcccgac tggccgaata tttgaatcaa 600 ggcaaacgaa ttacgttact atgcggcgcc ggctgtgcac aatctcatga attgctcatg 660 cagctatgcg acaagttaaa atcccccatg gtatccgctc tgcgaggcaa ggaatatctg 720 gagtatgaca acccttatta tgctggattg acgggactga tcgggtattc ttccgggtac 780 catgcgatga tggattgtga cgtcctgctt atgctcggaa cagacttccc ttacagacag 840 ttttaccctg aagatgcgat tgtcctacag gtagatatag agccagccca tctcggcaga 900 cgtactccgt tgacgtatgg tttatgcggg gatgtaaaag ccacattgga aatgctgcta 960 ccgcatttaa cgtcagagca tgattccaag catctagaga aaaccgtctc ccactatacc 1020 aaggtacgtc aggagttgga tgaccttgcc gttggtaaac caggtcatac gccgatccat 1080 ccccaatatc ttgccaaggt catcagtgac gccgcgcagg aaaatgctat tttcacttgt 1140 gatgtcggta ctcccactgt atgggcggcg cgttatttgc agatgaacgg tcagcgtcgg 1200 ctgctcggct cgttcaacca tggcacgatg gcaaatgcgc taccgcaggc aatcggagcg 1260 caggccactg agcctgaccg acaagtgatt gccctctcag gcgatggcgg gctcacgatg 1320 ctgatgggcg acctgctcac cctgaagcag catcagctgc ctattaaagt cattgttttc 1380 aataatggcg ctctcggttt tgtcgagctg gaaatgaaag cggccggatt cctggaaaac 1440 ggcactgaac tggtgaatcc tgattttggt gctgtagcgc aagctatggg actcaagggc 1500 atccgggttg aagatccgac catgctggag gatgccattc agcaagcatt ggctcatgat 1560 ggccctgttg tagtagacgt ggtggtgaac cgtcaagagc tatccatgcc acccaaaatt 1620 aatcttaaac aagcacaagg ctttacactg tggatgatga aagcgatgct gaacggacgc 1680 ggtgacgaga ttgttgaact ggctaagacc aatctctttc gttaa 1725 <210> 2 <211> 574 <212> PRT <213> Paenibacillus polymyxa <400> 2 Met Lys Thr Ile Ala Asp Thr Ile Val Gln Val Leu Val Asn Ala Gly   1 5 10 15 Val Lys Arg Ile Tyr Gly Ile Val Gly Asp Ser Leu Asn Asn Met Val              20 25 30 Asp Ser Ile Arg Ser Asn Gly Gln Ile Glu Trp Ile His Val Arg His          35 40 45 Glu Glu Val Ala Ala Phe Ala Ala Gly Ala Asp Ala Asp Leu Ser Gly      50 55 60 Ser Ile Ala Val Cys Ala Gly Ser Ser Gly Pro Gly Asn Leu His Leu  65 70 75 80 Ile Asn Gly Leu Tyr Asp Cys His Arg Asn Arg Val Pro Val Leu Ala                  85 90 95 Ile Ala Ala His Ile Pro Ser Asp Glu Ile Gly Ser Glu Tyr Phe Gln             100 105 110 Ala Thr His Pro Glu His Leu Phe Gly Glu Cys Ser His Phe Cys Glu         115 120 125 Val Ile Thr Thr Pro Arg Gln Ile Pro Arg Thr Val Thr Met Ala Ile     130 135 140 Gln Gln Ala Ile Ser Arg Ser Gly Val Ser Val Ile Val Leu Pro Gly 145 150 155 160 Asp Val Ala Leu Glu Ala Glu Lys Val Pro Ile Pro Glu His Val                 165 170 175 Tyr His Pro Thr Ala Pro Val Val His Ser Ser Ala Ser Glu Ile Ser             180 185 190 Arg Leu Ala Glu Tyr Leu Asn Gln Gly Lys Arg Ile Thr Leu Leu Cys         195 200 205 Gly Ala Gly Cys Ala Gln Ser His Glu Leu Leu Met Gln Leu Cys Asp     210 215 220 Lys Leu Lys Ser Pro Met Val Ser Ala Leu Arg Gly Lys Glu Tyr Leu 225 230 235 240 Glu Tyr Asp Asn Pro Tyr Tyr Ala Gly Leu Thr Gly Leu Ile Gly Tyr                 245 250 255 Ser Ser Gly Tyr His Ala Met Met Asp Cys Asp Val Leu Leu Met Leu             260 265 270 Gly Thr Asp Phe Pro Tyr Arg Gln Phe Tyr Pro Glu Asp Ala Ile Val         275 280 285 Leu Gln Val Asp Ile Glu Pro Ala His Leu Gly Arg Arg Thr Pro Leu     290 295 300 Thr Tyr Gly Leu Cys Gly Asp Val Lys Ala Thr Leu Glu Met Leu Leu 305 310 315 320 Pro His Leu Thr Ser Glu His Asp Ser Lys His Leu Glu Lys Thr Val                 325 330 335 Ser His Tyr Thr Lys Val Arg Gln Glu Leu Asp Asp Leu Ala Val Gly             340 345 350 Lys Pro Gly His Thr Pro Ile His Pro Gln Tyr Leu Ala Lys Val Ile         355 360 365 Ser Asp Ala Gln Glu Asn Ale Ile Phe Thr Cys Asp Val Gly Thr     370 375 380 Pro Thr Val Trp Ala Ala Arg Tyr Leu Gln Met Asn Gly Gln Arg Arg 385 390 395 400 Leu Leu Gly Ser Phe Asn His Gly Thr Met Ala Asn Ala Leu Pro Gln                 405 410 415 Ala Ile Gly Ala Gln Ala Thr Glu Pro Asp Arg Gln Val Ile Ala Leu             420 425 430 Ser Gly Asp Gly Gly Leu Thr Met Leu Met Gly Asp Leu Leu Thr Leu         435 440 445 Lys Gln His Gln Leu Pro Ile Lys Val Ile Val Phe Asn Asn Gly Ala     450 455 460 Leu Gly Phe Val Glu Leu Glu Met Lys Ala Glu Ply Leu Glu Asn 465 470 475 480 Gly Thr Glu Leu Val Asn Pro Asp Phe Gly Ala Val Ala Gln Ala Met                 485 490 495 Gly Leu Lys Gly Ile Arg Val Glu Asp Pro Thr Met Leu Glu Asp Ala             500 505 510 Ile Gln Gln Ala Leu Ala His Asp Gly Pro Val Val Val Asp Val Val         515 520 525 Val Asn Arg Gln Glu Leu Ser Met Pro Pro Lys Ile Asn Leu Lys Gln     530 535 540 Ala Gln Gly Phe Thr Leu Trp Met Met Lys Ala Met Leu Asn Gly Arg 545 550 555 560 Gly Asp Glu Ile Val Glu Leu Ala Lys Thr Asn Leu Phe Arg                 565 570 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> PoxB-hind primer <400> 3 aagcttctag acaggaggaa catctatg 28 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PoxB-bam primer <400> 4 ggatcctaac aggcaggacg aatggg 26 <210> 5 <211> 2503 <212> DNA <213> Artificial Sequence <220> <223> poxBpp-gfp fusion gene <400> 5 atgggtaccg ggcccatgaa gacaatcgca gatactattg tacaagtttt ggtcaatgca 60 ggggtcaagc ggatttatgg cattgttgga gactctctaa ataatatggt tgattccatt 120 cgcagcaatg gtcaaatcga atggattcat gtaaggcacg aagaagtggc tgcctttgca 180 gccggagcgg atgctgacct tagtggcagc attgctgtat gtgctggaag tagtggtccc 240 ggaaatttgc atctgattaa tggtttatat gattgccacc gcaatcgggt gcctgtactg 300 gctattgccg ctcatattcc aagcgacgaa atcggaagtg aatattttca agcgacgcat 360 cctgagcatc ttttcggaga atgcagtcac ttttgtgagg ttattacgac accgcgtcaa 420 attcccagaa cggtgaccat ggccattcaa caggcaattt cacgttcagg tgtctccgtc 480 attgttcttc ccggtgatgt agcagctttg gaggcggaaa aggtacccat ccctgaacat 540 gtctaccatc ccactgcacc tgtagtgcat ccgtcagctt ccgaaatttc ccgactggcc 600 gaatatttga atcaaggcaa acgaattacg ttactatgcg gcgccggctg tgcacaatct 660 catgaattgc tcatgcagct atgcgacaag ttaaaatccc ccatggtatc cgctctgcga 720 ggcaaggaat atctggagta tgacaaccct tattatgctg gattgacggg actgatcggg 780 tattcttccg ggtaccatgc gatgatggat tgtgacgtcc tgcttatgct cggaacagac 840 ttcccttaca gacagtttta ccctgaagat gcgattgtcc tacaggtaga tatagagcca 900 gcccatctcg gcagacgtac tccgttgacg tatggtttat gcggggatgt aaaagccaca 960 ttggaaatgc tgctaccgca tttaacgtca gagcatgatt ccaagcatct agagaaaacc 1020 gtctcccact ataccaaggt acgtcaggag ttggatgacc ttgccgttgg taaaccaggt 1080 catacgccga tccatcccca atatcttgcc aaggtcatca gtgacgccgc gcaggaaaat 1140 gctattttca cttgtgatgt cggtactccc actgtatggg cggcgcgtta tttgcagatg 1200 aacggtcagc gtcggctgct cggctcgttc aaccatggca cgatggcaaa tgcgctaccg 1260 caggcaatcg gagcgcaggc cactgagcct gaccgacaag tgattgccct ctcaggcgat 1320 ggcgggctcg cgatgctgat gggcgacctg ctcaccctga agcagcatca gctgcctatt 1380 aaagtcattg ttttcaataa tggcgctctc ggttttgtcg agctggaaat gaaagcggcc 1440 ggattcctgg aaaacggcac tgaactggtg aatcctgatt ttggtgctgt agcgcaagct 1500 atgggactca agggcatccg ggttgaagat ccgaccatgc tggaggatgc cattcagcaa 1560 gcattggctc atgatggccc tgttgtagta gacgtggtgg tgaaccgtca agagctatcc 1620 atgccaccca aaattaatct taaacaagca caaggcttta cactgtggat gatgaaagcg 1680 atgctgaacg gacgcggtga cgagattgtt gaactggcta agaccaatct ctttcgtctc 1740 gaggtcgacg gtatcgataa gcttgatatc gaattcctgc agatgagtaa aggagaagaa 1800 cttttcactg gagttgtccc aattcttgtt gaattagatg gtgacgttaa tgggcacaaa 1860 ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt 1920 atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac tctgacttat 1980 ggtgttcaat gcttttcaag atacccagat catatgaaac agcatgactt tttcaagagt 2040 gccatgcccg aaggttatgt acaggaaaga actatatttt tcaaagatga cgggaactac 2100 aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat cgagttaaaa 2160 ggtattgatt ttaaagaaga tggaaacatt cttggacaca aattggaata caactataac 2220 tcacacaatg tatacatcat ggcagacaaa caaaagaatg gaatcaaagt taacttcaaa 2280 attagacaca acattgaaga tggaagcgtt caactagcag accattatca acaaaatact 2340 ccaattggcg atggccctgt cctttcacca gacaaccatt acctgtccac acaatctgcc 2400 ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct 2460 gctgggatta cacatggcat ggatgaacta tacaaataaa tga 2503 <210> 6 <211> 832 <212> PRT <213> Artificial Sequence <220> <223> poxBpp-gfp fusion protein <400> 6 Met Gly Thr Gly Pro Met Lys Thr Ile Ala Asp Thr Ile Val Gln Val   1 5 10 15 Leu Val Asn Ala Gly Val Lys Arg Ile Tyr Gly Ile Val Gly Asp Ser              20 25 30 Leu Asn Asn Met Val Asp Ser Ile Arg Ser Asn Gly Gln Ile Glu Trp          35 40 45 Ile His Val Arg His Glu Glu Val Ala Ala Phe Ala Ala Gly Ala Asp      50 55 60 Ala Asp Leu Ser Gly Ser Ile Ala Val Cys Ala Gly Ser Ser Gly Pro  65 70 75 80 Gly Asn Leu His Leu Ile Asn Gly Leu Tyr Asp Cys His Arg Asn Arg                  85 90 95 Val Pro Val Leu Ala Ala Ala His Ile Pro Ser Asp Glu Ile Gly             100 105 110 Ser Glu Tyr Phe Gln Ala Thr His Pro Glu His Leu Phe Gly Glu Cys         115 120 125 Ser His Phe Cys Glu Val Ile Thr Thr Pro Arg Gln Ile Pro Arg Thr     130 135 140 Val Thr Met Ala Ile Gln Gln Ala Ile Ser Arg Ser Gly Val Val Ser 145 150 155 160 Ile Val Leu Pro Gly Asp Val Ala Ala Leu Glu Ala Glu Lys Val Pro                 165 170 175 Ile Pro Glu His Val Tyr His Pro Thr Ala Pro Val Val His Ser Ser             180 185 190 Ala Ser Glu Ile Ser Arg Leu Ala Glu Tyr Leu Asn Gln Gly Lys Arg         195 200 205 Ile Thr Leu Leu Cys Gly Ala Gly Cys Ala Gln Ser His Glu Leu Leu     210 215 220 Met Gln Leu Cys Asp Lys Leu Lys Ser Pro Met Val Ser Ala Leu Arg 225 230 235 240 Gly Lys Glu Tyr Leu Glu Tyr Asp Asn Pro Tyr Tyr Ala Gly Leu Thr                 245 250 255 Gly Leu Ile Gly Tyr Ser Ser Gly Tyr His Ala Met Met Asp Cys Asp             260 265 270 Val Leu Leu Met Leu Gly Thr Asp Phe Pro Tyr Arg Gln Phe Tyr Pro         275 280 285 Glu Asp Ala Ile Val Leu Gln Val Asp Ile Glu Pro Ala His Leu Gly     290 295 300 Arg Arg Thr Pro Leu Thr Tyr Gly Leu Cys Gly Asp Val Lys Ala Thr 305 310 315 320 Leu Glu Met Leu Leu Pro His Leu Thr Ser Glu His Asp Ser Lys His                 325 330 335 Leu Glu Lys Thr Val Ser His Tyr Thr Lys Val Arg Gln Glu Leu Asp             340 345 350 Asp Leu Ala Val Gly Lys Pro Gly His Thr Pro Ile His Pro Gln Tyr         355 360 365 Leu Ala Lys Val Ile Ser Asp Ala Gln Glu Asn Ala Ile Phe Thr     370 375 380 Cys Asp Val Gly Thr Pro Thr Val Trp Ala Ala Arg Tyr Leu Gln Met 385 390 395 400 Asn Gly Gln Arg Arg Leu Leu Gly Ser Phe Asn His Gly Thr Met Ala                 405 410 415 Asn Ala Leu Pro Gln Ala Ile Gly Ala Gln Ala Thr Glu Pro Asp Arg             420 425 430 Gln Val Ile Ala Leu Ser Gly Asp Gly Gly Leu Thr Met Leu Met Gly         435 440 445 Asp Leu Leu Thr Leu Lys Gln His Gln Leu Pro Ile Lys Val Ile Val     450 455 460 Phe Asn Asn Gly Ala Leu Gly Phe Val Glu Leu Glu Met Lys Ala Ala 465 470 475 480 Gly Phe Leu Glu Asn Gly Thr Glu Leu Val Asn Pro Asp Phe Gly Ala                 485 490 495 Val Ala Gln Ala Met Gly Leu Lys Gly Ile Arg Val Glu Asp Pro Thr             500 505 510 Met Leu Glu Asp Ala Ile Gln Gln Ala Leu Ala His Asp Gly Pro Val         515 520 525 Val Val Asp Val Val Val Asn Arg Gln Glu Leu Ser Met Pro Pro Lys     530 535 540 Ile Asn Leu Lys Gln Ala Gln Gly Phe Thr Leu Trp Met Met Lys Ala 545 550 555 560 Met Leu Asn Gly Arg Gly Asp Glu Ile Val Glu Leu Ala Lys Thr Asn                 565 570 575 Leu Phe Arg Leu Glu Val Asp Gly Ile Asp Lys Leu Asp Ile Glu Phe             580 585 590 Leu Gln Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile         595 600 605 Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Ser Ser     610 615 620 Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe 625 630 635 640 Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr                 645 650 655 Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met             660 665 670 Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln         675 680 685 Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala     690 695 700 Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys 705 710 715 720 Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu                 725 730 735 Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys             740 745 750 Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly         755 760 765 Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp     770 775 780 Gly Pro Val Leu Ser Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala 785 790 795 800 Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu                 805 810 815 Phe Val Thr Ala Gly Ily Thr Gly Met Asp Glu Leu Tyr Lys             820 825 830 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> pOXBF-apa primer <400> 7 aaagggccca tgaagacaat cgcagatac 29 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> pOXBR-xho primer <400> 8 atatctcgag acgaaagaga ttggtcttag 30 <210> 9 <211> 3597 <212> DNA <213> Artificial Sequence <220> <223> poxBpp-amyE fusion gene <400> 9 atgggtaccg ggcccatgaa gacaatcgca gatactattg tacaagtttt ggtcaatgca 60 ggggtcaagc ggatttatgg cattgttgga gactctctaa ataatatggt tgattccatt 120 cgcagcaatg gtcaaatcga atggattcat gtaaggcacg aagaagtggc tgcctttgca 180 gccggagcgg atgctgacct tagtggcagc attgctgtat gtgctggaag tagtggtccc 240 ggaaatttgc atctgattaa tggtttatat gattgccacc gcaatcgggt gcctgtactg 300 gctattgccg ctcatattcc aagcgacgaa atcggaagtg aatattttca agcgacgcat 360 cctgagcatc ttttcggaga atgcagtcac ttttgtgagg ttattacgac accgcgtcaa 420 attcccagaa cggtgaccat ggccattcaa caggcaattt cacgttcagg tgtctccgtc 480 attgttcttc ccggtgatgt agcagctttg gaggcggaaa aggtacccat ccctgaacat 540 gtctaccatc ccactgcacc tgtagtgcat ccgtcagctt ccgaaatttc ccgactggcc 600 gaatatttga atcaaggcaa acgaattacg ttactatgcg gcgccggctg tgcacaatct 660 catgaattgc tcatgcagct atgcgacaag ttaaaatccc ccatggtatc cgctctgcga 720 ggcaaggaat atctggagta tgacaaccct tattatgctg gattgacggg actgatcggg 780 tattcttccg ggtaccatgc gatgatggat tgtgacgtcc tgcttatgct cggaacagac 840 ttcccttaca gacagtttta ccctgaagat gcgattgtcc tacaggtaga tatagagcca 900 gcccatctcg gcagacgtac tccgttgacg tatggtttat gcggggatgt aaaagccaca 960 ttggaaatgc tgctaccgca tttaacgtca gagcatgatt ccaagcatct agagaaaacc 1020 gtctcccact ataccaaggt acgtcaggag ttggatgacc ttgccgttgg taaaccaggt 1080 catacgccga tccatcccca atatcttgcc aaggtcatca gtgacgccgc gcaggaaaat 1140 gctattttca cttgtgatgt cggtactccc actgtatggg cggcgcgtta tttgcagatg 1200 aacggtcagc gtcggctgct cggctcgttc aaccatggca cgatggcaaa tgcgctaccg 1260 caggcaatcg gagcgcaggc cactgagcct gaccgacaag tgattgccct ctcaggcgat 1320 ggcgggctcg cgatgctgat gggcgacctg ctcaccctga agcagcatca gctgcctatt 1380 aaagtcattg ttttcaataa tggcgctctc ggttttgtcg agctggaaat gaaagcggcc 1440 ggattcctgg aaaacggcac tgaactggtg aatcctgatt ttggtgctgt agcgcaagct 1500 atgggactca agggcatccg ggttgaagat ccgaccatgc tggaggatgc cattcagcaa 1560 gcattggctc atgatggccc tgttgtagta gacgtggtgg tgaaccgtca agagctatcc 1620 atgccaccca aaattaatct taaacaagca caaggcttta cactgtggat gatgaaagcg 1680 atgctgaacg gacgcggtga cgagattgtt gaactggcta agaccaatct ctttcgtctc 1740 gaggcaccgt cgatcaaaag cggaaccatt cttcatgcat ggaattggtc gttcaatacg 1800 ttaaaacaca atatgaagga tattcatgat gcaggatata cagccattca gacatctccg 1860 attaaccaag taaaggaagg gaatcaagga gataaaagca tgtcgaactg gtactggctg 1920 tatcagccga catcgtatca aattggcaac cgttacttag gtactgaaca agaatttaaa 1980 gaaatgtgtg cagccgctga agaatatggc ataaaggtca ttgttgacgc ggtcatcaat 2040 cataccacca gtgattatgc cgcgatttcc aatgaggtta agagtattcc aaactggaca 2100 catggaaaca cacaaattaa aaactggtct gatcgatggg atgtcacgca gaattcattg 2160 ctcgggctgt atgactggaa tacacaaaat acacaagtac agtcctatct gaaacggttc 2220 ttagacaggg cattgaatga cggggcagac ggttttcgat ttgatgccgc caaacatata 2280 gagcttccag atgatggcag ttacggcagt caattttggc cgaatatcac aaatacatct 2340 gcagagttcc aatacggaga aatcctgcag gatagtgcct ccagagatgc tgcatatgcg 2400 aattatatgg atgtgacagc gtctaactat gggcattcca taaggtccgc tttaaagaat 2460 cgtaatctgg gcgtgtcgaa tatctcccac tatgcatctg atgtgtctgc ggacaagcta 2520 gtgacatggg tagagtcgca tgatacgtat gccaatgatg atgaagagtc gacatggatg 2580 agcgatgatg atatccgttt aggctgggcg gtgatagctt ctcgttcagg cagtacgcct 2640 cttttctttt ccagacctga gggaggcgga aatggtgtga ggttcccggg gaaaagccaa 2700 ataggcgatc gcgggagtgc tttatttgaa gatcaggcta tcactgcggt caatagattt 2760 cacaatgtga tggctggaca gcctgaggaa ctctcgaacc cgaatggaaa caaccagata 2820 tttatgaatc agcgcggctc acatggcgtt gtgctggcaa atgcaggttc atcctctgtc 2880 tctatcaata cggcaacaaa attgcctgat ggcaggtatg acaataaagc tggagcgggt 2940 tcatttcaag tgaacgatgg taaactgaca ggcacgatca atgccaggtc tgtagctgtg 3000 ctttatcctg atgatattgc aaaagcgcct catgttttcc ttgagaatta caaaacaggt 3060 gtaacacatt ctttcaatga tcaactgacg attaccttgc gtgcagatgc gaatacaaca 3120 aaagccgttt atcaaatcaa taatggacca gacgacaggc gtttaaggat ggagatcaat 3180 tcacaatcgg aaaaggagat ccaatttggc aaaacataca ccatcatgtt aaaaggaacg 3240 aacagtgatg gtgtaacgag gaccgagaaa tacagttttg ttaaaagaga tccagcgtcg 3300 gccaaaacca tcggctatca aaatccgaat cattggagcc aggtaaatgc ttatatctat 3360 aaacatgatg ggagccgagt aattgaattg accggatctt ggcctggaaa accaatgact 3420 aaaaatgcag acggaattta cacgctgacg ctgcctgcgg acacggatac aaccaacgca 3480 aaagtgattt ttaataatgg cagcgcccaa gtgcccggtc agaatcagcc tggctttgat 3540 tacgtgctaa atggtttata taatgactcg ggcttaagcg gttctcttcc ccattga 3597 <210> 10 <211> 1198 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > PoxBpp-amyE fusion protein <400> 10 Met Gly Thr Gly Pro Met Lys Thr Ile Ala Asp Thr Ile Val Gln Val   1 5 10 15 Leu Val Asn Ala Gly Val Lys Arg Ile Tyr Gly Ile Val Gly Asp Ser              20 25 30 Leu Asn Asn Met Val Asp Ser Ile Arg Ser Asn Gly Gln Ile Glu Trp          35 40 45 Ile His Val Arg His Glu Glu Val Ala Ala Phe Ala Ala Gly Ala Asp      50 55 60 Ala Asp Leu Ser Gly Ser Ile Ala Val Cys Ala Gly Ser Ser Gly Pro  65 70 75 80 Gly Asn Leu His Leu Ile Asn Gly Leu Tyr Asp Cys His Arg Asn Arg                  85 90 95 Val Pro Val Leu Ala Ala Ala His Ile Pro Ser Asp Glu Ile Gly             100 105 110 Ser Glu Tyr Phe Gln Ala Thr His Pro Glu His Leu Phe Gly Glu Cys         115 120 125 Ser His Phe Cys Glu Val Ile Thr Thr Pro Arg Gln Ile Pro Arg Thr     130 135 140 Val Thr Met Ala Ile Gln Gln Ala Ile Ser Arg Ser Gly Val Val Ser 145 150 155 160 Ile Val Leu Pro Gly Asp Val Ala Ala Leu Glu Ala Glu Lys Val Pro                 165 170 175 Ile Pro Glu His Val Tyr His Pro Thr Ala Pro Val Val His Ser Ser             180 185 190 Ala Ser Glu Ile Ser Arg Leu Ala Glu Tyr Leu Asn Gln Gly Lys Arg         195 200 205 Ile Thr Leu Leu Cys Gly Ala Gly Cys Ala Gln Ser His Glu Leu Leu     210 215 220 Met Gln Leu Cys Asp Lys Leu Lys Ser Pro Met Val Ser Ala Leu Arg 225 230 235 240 Gly Lys Glu Tyr Leu Glu Tyr Asp Asn Pro Tyr Tyr Ala Gly Leu Thr                 245 250 255 Gly Leu Ile Gly Tyr Ser Ser Gly Tyr His Ala Met Met Asp Cys Asp             260 265 270 Val Leu Leu Met Leu Gly Thr Asp Phe Pro Tyr Arg Gln Phe Tyr Pro         275 280 285 Glu Asp Ala Ile Val Leu Gln Val Asp Ile Glu Pro Ala His Leu Gly     290 295 300 Arg Arg Thr Pro Leu Thr Tyr Gly Leu Cys Gly Asp Val Lys Ala Thr 305 310 315 320 Leu Glu Met Leu Leu Pro His Leu Thr Ser Glu His Asp Ser Lys His                 325 330 335 Leu Glu Lys Thr Val Ser His Tyr Thr Lys Val Arg Gln Glu Leu Asp             340 345 350 Asp Leu Ala Val Gly Lys Pro Gly His Thr Pro Ile His Pro Gln Tyr         355 360 365 Leu Ala Lys Val Ile Ser Asp Ala Gln Glu Asn Ala Ile Phe Thr     370 375 380 Cys Asp Val Gly Thr Pro Thr Val Trp Ala Ala Arg Tyr Leu Gln Met 385 390 395 400 Asn Gly Gln Arg Arg Leu Leu Gly Ser Phe Asn His Gly Thr Met Ala                 405 410 415 Asn Ala Leu Pro Gln Ala Ile Gly Ala Gln Ala Thr Glu Pro Asp Arg             420 425 430 Gln Val Ile Ala Leu Ser Gly Asp Gly Gly Leu Thr Met Leu Met Gly         435 440 445 Asp Leu Leu Thr Leu Lys Gln His Gln Leu Pro Ile Lys Val Ile Val     450 455 460 Phe Asn Asn Gly Ala Leu Gly Phe Val Glu Leu Glu Met Lys Ala Ala 465 470 475 480 Gly Phe Leu Glu Asn Gly Thr Glu Leu Val Asn Pro Asp Phe Gly Ala                 485 490 495 Val Ala Gln Ala Met Gly Leu Lys Gly Ile Arg Val Glu Asp Pro Thr             500 505 510 Met Leu Glu Asp Ala Ile Gln Gln Ala Leu Ala His Asp Gly Pro Val         515 520 525 Val Val Asp Val Val Val Asn Arg Gln Glu Leu Ser Met Pro Pro Lys     530 535 540 Ile Asn Leu Lys Gln Ala Gln Gly Phe Thr Leu Trp Met Met Lys Ala 545 550 555 560 Met Leu Asn Gly Arg Gly Asp Glu Ile Val Glu Leu Ala Lys Thr Asn                 565 570 575 Leu Phe Arg Leu Glu Ala Pro Ser Ile Lys Ser Gly Thr Ile Leu His             580 585 590 Ala Trp Asn Trp Ser Phe Asn Thr Leu Lys His Asn Met Lys Asp Ile         595 600 605 His Asp Ala Gly Tyr Thr Ala Ile Gln Thr Ser Pro Ile Asn Gln Val     610 615 620 Lys Glu Gly Asn Gln Gly Asp Lys Ser Ser Ser Asn Trp Tyr Trp Leu 625 630 635 640 Tyr Gln Pro Thr Ser Tyr Gln Ile Gly Asn Arg Tyr Leu Gly Thr Glu                 645 650 655 Gln Glu Phe Lys Glu Met Cys Ala Ala Ala Glu Glu Tyr Gly Ile Lys             660 665 670 Val Ile Val Asp Ala Val Ile Asn His Thr Thr Ser Asp Tyr Ala Ala         675 680 685 Ile Ser Asn Glu Val Lys Ser Ile Pro Asn Trp Thr His Gly Asn Thr     690 695 700 Gln Ile Lys Asn Trp Ser Asp Arg Trp Asp Val Thr Gln Asn Ser Leu 705 710 715 720 Leu Gly Leu Tyr Asp Trp Asn Thr Gln Asn Thr Gln Val Gln Ser Tyr                 725 730 735 Leu Lys Arg Phe Leu Asp Arg Ala Leu Asn Asp Gly Ala Asp Gly Phe             740 745 750 Arg Phe Asp Ala Lys His Ile Glu Leu Pro Asp Asp Gly Ser Tyr         755 760 765 Gly Ser Gln Phe Trp Pro Asn Ile Thr Asn Thr Ser Ala Glu Phe Gln     770 775 780 Tyr Gly Glu Ile Leu Gln Asp Ser Ala Ser Arg Asp Ala Ala Tyr Ala 785 790 795 800 Asn Tyr Met Asp Val Thr Ala Ser Asn Tyr Gly His Ser Ile Arg Ser                 805 810 815 Ala Leu Lys Asn Arg Asn Leu Gly Val Ser Asn Ile Ser His Tyr Ala             820 825 830 Ser Asp Val Ser Ala Asp Lys Leu Val Thr Trp Val Glu Ser His Asp         835 840 845 Thr Tyr Ala Asn Asp Asp Glu Glu Ser Thr Trp Met Ser Asp Asp Asp     850 855 860 Ile Arg Leu Gly Trp Ala Val Ile Ala Ser Arg Ser Ser Ser Thr Pro 865 870 875 880 Leu Phe Ser Arg Pro Glu Gly Gly Gly Asn Gly Val Arg Phe Pro                 885 890 895 Gly Lys Ser Gln Ile Gly Asp Arg Gly Ser Ala Leu Phe Glu Asp Gln             900 905 910 Ala Ile Thr Ala Val Asn Arg Phe His Asn Val Ala Gly Gln Pro         915 920 925 Glu Glu Leu Ser Asn Pro Asn Gly Asn Asn Gln Ile Phe Met Asn Gln     930 935 940 Arg Gly Ser His Gly Val Val Leu Ala Asn Ala Gly Ser Ser Ser Val 945 950 955 960 Ser Ile Asn Thr Ala Thr Lys Leu Pro Asp Gly Arg Tyr Asp Asn Lys                 965 970 975 Ala Gly Ala Gly Ser Phe Gln Val Asn Asp Gly Lys Leu Thr Gly Thr             980 985 990 Ile Asn Ala Arg Ser Val Ala Val Leu Tyr Pro Asp Asp Ile Ala Lys         995 1000 1005 Ala Pro His Val Phe Leu Glu Asn Tyr Lys Thr Gly Val Thr His Ser    1010 1015 1020 Phe Asn Asp Gln Leu Thr Ile Thr Leu Arg Ala Asp Ala Asn Thr Thr 1025 1030 1035 1040 Lys Ala Val Tyr Gln Ile Asn Asn Gly Pro Asp Asp Arg Arg Leu Arg                1045 1050 1055 Met Glu Ile Asn Ser Gln Ser Glu Lys Glu Ile Gln Phe Gly Lys Thr            1060 1065 1070 Tyr Thr Ile Met Leu Lys Gly Thr Asn Ser Asp Gly Val Thr Arg Thr        1075 1080 1085 Glu Lys Tyr Ser Phe Val Lys Arg Asp Pro Ala Ser Ala Lys Thr Ile    1090 1095 1100 Gly Tyr Gln Asn Pro Asn His Trp Ser Gln Val Asn Ala Tyr Ile Tyr 1105 1110 1115 1120 Lys His Asp Gly Ser Arg Val Ile Glu Leu Thr Gly Ser Trp Pro Gly                1125 1130 1135 Lys Pro Met Thr Lys Asn Ala Asp Gly Ile Tyr Thr Leu Thr Leu Pro            1140 1145 1150 Ala Asp Thr Asp Thr Thr Asn Ala Lys Val Ile Phe Asn Asn Gly Ser        1155 1160 1165 Ala Gln Val Pro Gly Gln Asn Gln Pro Gly Phe Asp Tyr Val Leu Asn    1170 1175 1180 Gly Leu Tyr Asn Asp Ser Gly Leu Ser Gly Ser Leu Pro His 1185 1190 1195 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AmyF-Xho primer <400> 11 atatctcgag gcaccgtcga tcaaaagcgg 30 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> amyR-spe primer <400> 12 attactagtt gtgccaacac ttcacaaac 29

Claims (13)

A fusion gene comprising a PoxB gene consisting of the nucleotide sequence of SEQ ID NO: 1 and a gene encoding a target protein linked to produce an inclusion body of the target protein.
The fusion gene according to claim 1, wherein the PoxB gene is derived from a Paenibacillus polymyxa.
delete 2. The fusion gene according to claim 1, wherein the target protein is green fluorescent protein (GFP) or amylase.
5. The fusion gene according to claim 4, wherein the fusion gene has the nucleotide sequence of SEQ ID NO: 5 or 9.
A fusion protein encoded by the fusion gene of any one of claims 1, 2, 4, or 5.
7. The fusion protein of claim 6, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 6 or 10.
An expression vector comprising the fusion gene of any one of claims 1, 2, 4, or 5.
A transformant in which the expression vector of claim 8 is transformed into a host cell.
10. The transformant according to claim 9, wherein the host cell is Escherichia coli.
(a) preparing a fusion gene by binding a PoxB gene comprising the nucleotide sequence of SEQ ID NO: 1 to a gene encoding a target protein to produce an active inclusion body;
(b) preparing an expression vector comprising the fusion gene;
(c) preparing a transformant transformed with the expression vector prepared in the host cell;
(d) culturing the transformant to produce an active inclusion body of the target protein.
12. The method of claim 11, further comprising: (e) cleaving the target protein in the produced active inclusion body.
12. The method of claim 11, wherein the target protein is GFP or amylase.

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