KR20200130093A - Auto-induction Plasmid - Google Patents

Abstract

The present invention relates to an auto-induction plasmid which simplifies a protein production process, does not require use of special modifications, and is efficient and economical. In the plasmid of the present invention, a gene for encoding a target protein is operatively linked to a promoter.

Description

Auto-induction Plasmid

The present invention relates to an efficient and economical autoinducible plasmid that simplifies the protein production process and does not require the use of special modifications.

Bacillus, along with E. coli and yeast, has been used as a major host for producing industrially useful proteins. The advantages of Bacillus as a protein production host are: (1) harmless to the human body, (2) able to secrete large amounts of protein outside the cell, (3) not producing endotoxins, and (4) GRAS by FDA It is defined as (generally regared as safe), (5) there is no codon bias, (6) genetic manipulation, transformation and mass culture are easy, and (7) genomic information is known. Currently, about 60% of the world's industrial enzymes use Bacillus as a host bacteria, and it is known to be suitable for the production of proteins for food or medicine due to its nature as a generally regarded as safe (GRAS) microorganism that is harmless to the human body.

Traditionally, in order to produce useful enzymes or proteins in large quantities, methods of mutating host bacteria through treatment with mutagenesis, ultraviolet or irradiation, etc. have been used, but these methods often result in the growth rate of host bacteria due to nonselective mutations. Is reduced, or it is difficult to optimize the medium composition and culture conditions. In addition, in the case of mutant strains, strain degeneration occurs, and since host bacteria for producing each target protein must be developed separately, it is a classic method that takes a lot of time and effort. In recent years, due to the development of genetic engineering, various expression systems have been developed, and production of useful proteins has been attempted. According to this report, a method for artificially inducing expression using specific expression inducing factors such as IPTG and sugar, a method using a cell growth cycle dependent promoter, and automatic induction expression have been developed. However, several problems remain, such as the unit price of the expression inducing factor affects productivity, a large amount of protein is expressed during cell growth, affects cell growth, or increases the production cost to maintain low temperature conditions. In addition, although the above-described expression systems have been developed, there is no example of successful market entry of protein drugs produced by Bacillus as a host bacteria, because (1) there is still no strong and controllable promoter compared to E. coli, (2 ) There is a problem in the stability of the expressed protein due to the presence of many proteolytic enzymes, and (3) it is interpreted that the decomposition by proteolytic enzymes present in the cell membrane occurs when the expressed protein is secreted outside the cell.

In the case of Bacillus, an expression system using a promoter of Bacillus certilus proteolytic enzyme 　aprE, a system using two types of promoters in parallel, and a system using a promoter of Bacillus thuringiensis 　cryIIIA have been developed. In these systems, protein expression proceeds even during the cell growth phase, so that when mass expression of a target protein is expressed, it is difficult to express a protein that affects cell growth or exhibits toxicity to cells. Meanwhile, Bacillus certilis 　 pstS 　 promoter has been used to develop a system capable of specifically expressing the cell growth arrest phase.

On the other hand, in the case of Escherichia coli, which is widely used as an expression host, since most of the expressed proteins are present in cells, a physical, chemical or enzymatic cell destruction method is required. Therefore, in order to mass-produce a protein of interest, an inexpensive method for cell destruction is required. In the case of Bacillus, at the end of the cell growth arrest period, the cells are automatically destroyed and the proteins in the cells are exposed to the medium, and this feature enables efficient recovery of the proteins expressed in the cells.

Korean Patent Publication No. 10-2007-0053765

An object of the present invention is to provide an efficient and economical autoinducible plasmid that simplifies the protein production process and does not require the use of special modifications.

1. Recombinant plasmid containing a promoter gene consisting of the sequence of SEQ ID NO: 1.

2. The plasmid according to 1 above, wherein the gene encoding the protein of interest is operatively linked with the promoter.

3. The plasmid according to the above 1, wherein the plasmid has a promoter gene having an operator substituted with a promoter gene consisting of the sequence of SEQ ID NO: 1.

4. A host microorganism transformed with a recombinant plasmid containing a promoter gene consisting of the sequence of SEQ ID NO: 1.

5. The microorganism according to 4 above, wherein the gene encoding the target protein is operatively linked with the promoter.

6. The microorganism according to the above 4, wherein the plasmid has a promoter gene having an operator substituted with a promoter gene consisting of the sequence of SEQ ID NO: 1.

7. The microorganism according to the above 4, wherein the microorganism is at least one strain selected from the group consisting of E. coli, Pseudomonas, cyanobacteria, and Bacillus.

8. A composition for producing a protein containing the plasmid or microorganism of any one of the above 1 to 7.

9. A method of producing a protein of interest comprising culturing the microorganism of any one of the above 4 to 7.

10. In the above 9, the production method further comprising the step of recovering the target protein.

11. The production method according to the above 9, wherein the culture is made in TB medium.

The auto-inducible plasmid of the present invention simplifies the protein production process and does not require the use of special modifications, thereby being very efficient and economical.

1 is a schematic diagram of the plasmid of the present invention (pVP65KR-gab-SacB(-)).
FIG. 2 is a photograph showing the protein production level of Rosetta2(DE3)pLysS/pVP65KR-SacB(-) grown under IPTG induction in LB (Luria-Bertani) medium.
3 is a photograph showing the degree of protein production of XL10-Gold/pVP65KR-gab-SacB(-) grown in TB (Terrific Broth) medium.

Hereinafter, the present invention will be described in detail.

The present invention provides a recombinant plasmid comprising a promoter gene consisting of the sequence of SEQ ID NO: 1.

In the present invention, the term "promoter" refers to an untranslated nucleic acid sequence upstream of the coding region that includes a binding site for a polymerase and has an activity to initiate transcription of a lower gene of a promoter into mRNA.

The promoter gene sequence of the present invention can be modified to a certain degree. Those skilled in the art know that the nucleotide sequence in which 70% or more homology is maintained by such artificial modification is equivalent to that derived from the nucleotide sequence of the present invention as long as it retains the promoter activity for expression of the gene of interest in the present invention. It will be easy to understand.

In the present invention, the term "homology" refers to the degree of homology with the nucleic acid sequence of SEQ ID NO: 1, and the comparison of homology is performed with the naked eye or by using an easy-to-purchase comparison program. Can be calculated as A nucleic acid sequence identical to the sequence of SEQ ID NO: 1 of the present invention is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more.

In addition, the promoter of the present invention includes variants having a promoter nucleic acid sequence in which one or more nucleic acid bases are mutated by substitution, deletion, insertion, or a combination thereof, as long as it retains promoter activity. Through such sequence variation, the same activity as the promoter of the present invention may be exhibited, but the function of the promoter can be improved suitably for a purpose, such as a promoter with increased activity. The mutation can be made by various methods known in the field of the present invention, examples of which include error-prone PCR, DNA shuffling, and site-directed mutagenesis.

Promoter genes of all categories are provided as promoter components of expression vectors for inducing expression of a gene of interest, and modifications of various plasmids using the promoter are included in the scope of the present invention.

In the present invention, the term "plasmid" refers to a gene construct including essential regulatory elements such as a promoter so that the gene of interest can be expressed in a suitable host, and may be a form integrated into the genome of a host cell or microorganism. .

In the present invention, "operably linked" refers to a functionally linked nucleotide sequence encoding a protein of interest with a promoter or variant nucleic acid sequence thereof of the present invention to perform a general function. The operative linkage with the recombinant plasmid can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage use enzymes generally known in the art.

In the present invention, the term "regulatory element" refers to an untranslated nucleic acid sequence that helps or affects the transcription, translation, or expression of a nucleic acid sequence encoding a protein. The plasmid of the present invention essentially contains a promoter or a variant thereof of the present invention as a regulatory element, and an expression control sequence capable of affecting the expression of a protein, for example, an initiation codon, a stop codon, a polyadenylation signal, an enhancer , A signal sequence for membrane targeting or secretion, and the like.

In addition, in the case of a replicable expression plasmid, the plasmid may include a replication origin, which is a specific nucleic acid sequence from which replication is initiated.

In addition, the plasmid may include a selection marker. The selectable marker is for selecting cells or microorganisms transformed with a plasmid, and markers that confer a selectable phenotype such as drug resistance, nutritional demand, resistance to cytotoxic agents, or expression of surface proteins may be used. Since only cells or microorganisms expressing a selection marker survive in an environment treated with a selective agent, a transformed individual can be selected.

The plasmid of the present invention may be one in which a gene encoding a protein of interest is operatively linked to the promoter, and specifically, may be linked to a lower region of the promoter.

As a gene encoding the protein of interest, the protein of interest that is useful medically and industrially may include hormones, cytokines, enzymes, coagulation factors, transport proteins, receptors, regulatory proteins, structural proteins, transcription factors, antigens, antibodies, etc., specifically , Gene encoding MBP (Maltose Binding Protein), gene encoding mCherry protein, factor VIII, Christmas factor IX, tissue plasminogen activator, insulin, Glucagon, growth hormone, gonadotrophin, erythropoietin, colony stimulating factor, interferon, interleukin, vaccine antigen ), a monoclonal antibody, and a tumor necrosis factor, but may be a gene encoding at least one selected from the group consisting of, but is not limited thereto.

The promoter gene may be one in which activity is induced in the host growth stationary phase, which is, in the growth cycle of cells or microorganisms transformed with the plasmid of the present invention, host growth arrests reaching the maximum cell division rate. Protein production can be automatically produced without adding special protein production initiating substances, such as IPTG, lactose, etc., on the medium to the stationary-phase, and an additional transformation step with a separate microorganism for protein production is required. It is possible to produce proteins with excellent efficiency even without passing through, which is very economical and may be the main cause of high utility value.

The plasmid of the present invention may use a vector skeleton selected from the group consisting of pVP, pQE, pET, pMAL, pGEX and pGEM as a basic skeleton, but is not particularly limited thereto, and a vector capable of recombining the promoter sequence of the present invention If it is a skeleton, any vector skeleton well known in the art is not particularly limited.

When using the vector backbone known in the art as the basic backbone of the plasmid of the present invention, the existing promoter including its operator may be substituted with the promoter of the present invention. In this case, the vector It can be substituted by the process of replacing the promoter with the promoter of the present invention using a pair of primers that complementarily bind near the promoter and the operator gene sequence of the backbone.

As a specific example of the substitution, when using pVP (pVP65K) as a vector backbone well known in the art, its basic promoter and operator T5 promoter and lac operator may be substituted with the promoter of the present invention, In this case, the promoter of the present invention can be substituted by using a pair of primers each consisting of the sequences of SEQ ID NOs: 2 and 3.

The present invention provides a host microorganism transformed with the plasmid described above.

There is no particular limitation on the host microorganism, which is a characteristic of the promoter of the present invention, and any strain including the plasmid of the present invention can produce a target protein with excellent efficiency, regardless of strain.

As a specific example of the host microorganism, it may be at least one selected from the group consisting of E. coli, Pseudomonas, cyanobacteria, and Bacillus, but as described above, It is not.

The transformation may be performed by a method conventional in the art, and may be introduced through, for example, a natural introduction method, a heat shock method, an electric shock method, etc., but is not particularly limited thereto.

Specific details related to the plasmid are as described above.

The present invention provides a composition for producing a protein comprising the plasmid or microorganism.

The composition of the present invention may additionally contain an auxiliary material for producing a protein of interest with excellent efficiency, and for specific examples, it further includes at least one weak stress treatment material selected from the group consisting of salt, ethanol, sorbitol, and aqueous hydrogen peroxide. However, it is not necessarily limited thereto.

Specific details related to the plasmid and microorganism are as described above.

The present invention provides a method for producing a target protein comprising culturing the above-described microorganism.

In addition, the production method of the present invention may further include the step of recovering the target protein, and this recovery is associated with a conventional process in the art to use the produced protein for a purpose, such as an additional protein purification process. Can be done.

In addition, in the culture, it is not limited to a specific medium, but preferably contains glycerol, so that the persistence of cell growth is high, and the cell growth effect is excellent compared to the culture medium of the eastern coat. It is presented as specific data in the following examples.

As described above, the production method of the present invention can automatically produce proteins without adding a special protein production initiating substance, such as IPTG, lactose, etc., on a separate medium, and additionally to a separate microorganism for protein production. It has a very economical advantage because it is possible to produce proteins with excellent efficiency without going through the transformation step.

The production method of the present invention further comprises culturing at low temperature to control the strength of the promoter, or by adding at least one weak stress treatment material selected from the group consisting of salt, ethanol, sorbitol and hydrogen peroxide to change the strength of the promoter. Accordingly, it may further include the step of controlling the production level of the target protein.

Specific matters related to the microorganism are as described above.

Hereinafter, examples will be described in detail to illustrate the present invention in detail.

Experiment method

1. Vectors, genes, oligonucleotides and kits

The pVP65K vector was obtained from the Eukaryotic Structural Genomics (http://www.uwstructuralgenomics.org/) center, and the pVP80K vector was purchased from DNASU (http://www.dnasu.org). The MBP-mCherry-8xHis gene block was synthesized by Integrated DNA Technologies (http://www.idtdna.com). The QuikChange Lightning Site-Directed Mutagenesis kit was purchased from Agilent (http://www.agilent.com). All oligonucleotides are Cosmogenetech co, Ltd. (http://www.cosmogenetech.com) synthesized by.

2. E. coli strain

Rosetta2(DE3) pLysS was purchased from Merck KGaA (http://www.merckmillipore.com) and XL10-Gold was included in the QuikChange Lightning Site-Directed Mutagenesis kit.

3. Plasmid design

The pVP65K vector (SEQ ID NO: 8) was used as an initial template. The MBP-mCherry-8xHis gene block (SEQ ID NO: 11) was digested with NcoI and NotI, and ligated with pVP65K previously digested with the same restriction enzyme to replace the original MBP (Maltose Binding Protein) gene. The three gene fragments of this gene block share the same reading frame with no internal stop codons, resulting in a single fusion protein capable of attaching the target protein right next to it. The resulting plasmid was named pVP65KR, and indicated by "R" indicating red color of mCherry (a variant of red fluorescent protein). Subsequently, the portion containing the barnase gene (SEQ ID NO: 12) of pVP65KR was replaced with a levansucrase (SacB) gene (SEQ ID NO: 13). The levan sucrase gene was amplified by PCR using two oligonucleotides each consisting of the sequences of SEQ ID NOs: 4 and 5 from the pVP80K vector. The resulting PCR product was purified, digested with SgfI and SacI, and ligated with pVP65KR digested with the same restriction enzyme set previously. The resulting plasmid was named pVP65KR-SacB. The HindIII site inside the SacB gene was removed using a QuikChange Lightning Site-Directed Mutagenesis kit having two mutant primer pairs each consisting of the sequences of SEQ ID NOs: 6 and 7. The resulting plasmid was named pVP65KR-SacB(-) ((-) indicates the removal of the native HindIII). The T5 promoter (T5 promoter, SEQ ID NO: 9) and the lac operator (SEQ ID NO: 10) of pVP65KR-SacB(-) are QuikChange Lightning Site-Directed, each having a primer pair consisting of the sequences of SEQ ID NOs: 2 and 3 Using the Mutagenesis kit, it was substituted with a hybrid stationary phase promoter consisting of the sequence of SEQ ID NO: 1. The resulting plasmid was named pVP65KR-gab-SacB(-).

4. Culture of E. coli

(1) mCherry production comparison experiment

A fully grown LB culture solution of Rosetta2(DE3) pLysS containing pVP65KR-SacB(-) was inoculated in 50 mL of LB medium. Incubation was performed at 37°C with a shaking incubator. IPTG was added to a final concentration of 0.5 mM when an OD of 600 mM reached 0.8, and the culture was harvested after 3 hours. The harvested cells were resuspended in 10 mL of Tris HCl pH 8.0.

50 μL of XL10-Gold LB culture medium with pVP65KR-SacB(-) was inoculated in 50 mL of LB or TB medium, and grown in a shaking incubator at 37°C for 18 hours.

50 mL LB cultures of Rosetta2(DE3) pLysS cells were grown separately overnight in 50 mL of LB medium, mixed with XL10-Gold cultures, and harvested in the same centrifuge tube. The harvested cells were resuspended in 10 mL of Tris HCl pH 8.0.

(2) MBP production comparison experiment

50 μL of fully grown LB culture solution of Rosetta2(DE3) pLysS containing pVP65KR-SacB(-) was inoculated in 50 mL of OvernightExpress® LB medium. It was grown in a shaking incubator at 37° C. for 18 hours. Cells harvested by centrifuge were resuspended in 10 mL of Tris HCl pH 8.0.

50 mL of TB medium was inoculated with a fully grown LB culture solution of Rosetta2(DE3) pLysS containing pVP65KR-SacB(-). Incubation was performed at 37°C with a shaking incubator. Cells harvested by centrifuge were resuspended in 10 mL of Tris HCl pH 8.0.

50 μL of a fully grown LB culture medium of XL10-Gold with pVP65KR-gad-SacB(-) was inoculated in 50 mL of TB medium, and grown in a shaking incubator at 37°C for 18 hours.

50 mL LB cultures of Rosetta2(DE3) pLysS cells were grown separately overnight in 50 mL of LB medium, mixed with XL10-Gold cultures, and harvested in the same centrifuge tube. The harvested cells were resuspended in 10 mL of Tris HCl pH 8.0.

5. Cell Lysis and Protein Quantification

The production of mCherry protein was monitored qualitatively by the intensity of red. After harvesting, cells were lysed by freezing and thawing. 1 mg of DNaseI was added to eliminate stickiness caused by DNA molecules. Triton X-100 was added to a final concentration of 1%, and the cell lysate was centrifuged at 20,000 g for 30 minutes at 4°C. 1 mL of the supernatant was used to measure the absorbance at 587 nm with a UV spectrophotometer. In order to calculate the concentration of mCherry protein in the supernatant, an extinction coefficient of 72000 M ^-1 · cm ^-1 was used.

MBP was purified and quantified by column chromatography. Cells cultured in 50 mL medium were harvested, resuspended in 10 mL buffer solution, and lysed by freezing and thawing. 1 mg of DNaseI was added to remove stickiness due to DNA molecules, and Triton X-100 was added to a final concentration of 1%. The cell lysate was centrifuged at 20,000 g for 30 minutes at 4° C. to separate the supernatant. The supernatant was loaded on the HisTrap column and washed with 40 mL of NPI-10 solution (10mM sodium phosphate pH 7.0, 300mM NaCl, 10mM imidazole), and the composition ratio of NPI-500 (10mM sodium phosphate pH 7.0, 300mM NaCl, 500mM imidazole) MBP was eluted while gradually increasing. MBP was eluted at about 30% NPI-500, and the elution volume was in the range of 12-15 mL. To calculate the concentration of MBP protein, an extinction coefficient of 66350 M ^-1 ·cm ^-1 at 280 nm was used.

The absorbance was measured at 280 nm and converted into mass, and the absorbance was measured at 600 nm and converted into the amount of cultured cells.

Experiment result

1. Plasmid design

The synthesized gene block sequentially contained genes for MBP, mCherry protein and eight histidine stretches (8xHis). 8xHis should be attached to the N-terminus of the target protein to simplify the purification process. The stop codons of the MBP and mCherry genes were removed, resulting in uninterrupted translation, producing one single fusion protein to be cleaved into the target protein by the TVMV protease. The cleavage site of the TVMV protease is between mCherry and 8xHis. The TVMV protease was produced from a coognate gene present upstream of the same plasmid. Therefore, since the one-to-one ratio between the target protein and the MBP-mCherry fragment is maintained, the amount of the resulting (soluble + insoluble) target protein can be quantified by measuring the absorbance at 587 nm of the cell lysate. To facilitate the subclonal process, we decided to keep the lethal gene, because the surviving colonies almost guaranteed the correct insertion of the target gene, but we did a levan to barnase. They preferred the sucrase (SacB) gene. The latter appears to be more effective in the selection of correctly constructed plasmids, but must be used with a special strain such as BR610 that can inhibit its activity. The SacB gene is compatible with many widely used strains because its lethality depends on the presence of sucrose. For the preparation of the plasmid itself, it can be selected by kanamycin only if sucrose is additionally used in the selection of the correct plasmid.

2. Comparison of mCherry production results

(1) Cultivation of E. coli

T7 lysozyme was produced in Rosetta2 (DE3) pLysS at the basal level, but was sufficient to lyse the harvested cells simply by freezing and thawing. We used this strain to mix XL10-Gold cells at the harvest stage, resuspend in buffer, then freeze and thaw to lyse the protein. A small amount of DNaseI was effective in breaking the DNA strands, and Triton X-100 was useful in making the supernatant transparent enough to be analyzed with a UV spectrophotometer. We used a typical induction procedure to add IPTG in the mid-log stage in LB to compare the effect of the present self-inducing plasmid on TB. We compared the overall yield of mCherry protein using the same amount of LB or TB cultures. As can be seen in Table 1 below, the final number of cells in TB is at least three times that of cells obtained from LB. The host strains Rosetta2 (DE3) pLysS and XL10-Gold both produced very similar OD600s in LB.

(2) protein quantification

Absorbance was measured using the centrifuged cell lysate. At harvest, the LB cell pellet has a more intense red color than in TB, but the latter becomes more intense after resuspending in the same amount of buffer. The absorbance was measured at 587 nm, the maximum absorption of mCherry. We averaged the three media grown independently and summarized the details in Table 1 below. Referring to Table 1 below, cells grew more in TB than in LB. Red was less intense in TB than in LB, but the total amount of red was greater in TB than in LB. Cells grew more in TB, but IPTG induction in TB produced more or less protein than LB. Interestingly, XL10-Gold with an autoinducing plasmid in LB produced as many proteins as the original plasmid and IPTG induction. In fact, we tried YT, 2xYT and SOC media, but none of these produced more protein than in TB. Therefore, we concluded that TB is the most suitable growth medium for the self-inducing plasmid of the present invention.

Plasmid Host strain Medium Induction method Average OD ₆₀₀ Average mCherry concentration (mg/L) pVP65KR-SacB(-) Rosetta2(DE3)pLysS LB IPTG 2.6 ± 0.17 63 ± 16 pVP65KR-gab-SacB(-) XL10-Gold TB None 8.9 ± 0.54 160 ± 47

The autoinducible plasmid (or vector) of the present invention not only simplifies the protein production process, but also does not require the use of special modifications such as Rosetta2 (DE3). An additional and practical advantage of the plasmid of the present invention is that the target protein gene is expressed at the same level, although it does not guarantee the production of a soluble or natural protein, as can be seen due to the expression of the mCherry gene and the color change to intense pink.

3. MBP production comparison experiment results

The experimental results repeated three times for each of the three culture conditions are shown in Table 2. The result of averaged MBP production per OD in consideration of the amount of cells was 0.46 mg in Comparative Example 1, 0.33 mg in Comparative Example 2, and 0.53 mg in Example 1.

Therefore, it was confirmed that the production of MBP per cell was the highest in the auto-induction plasmid of the present invention, and the cost-effectiveness ratio was also high because no special host or medium was used.

Sample Plasmid Host strain Medium Induction method OD ₆₀₀ Average MBP
(mg) Comparative Example 1 pVP65KR-SacB(-) Rosetta2(DE3)pLysS LB IPTG 10.34 0.46 10.56 10.36 Comparative Example 2 pVP65KR-SacB(-) Rosetta2(DE3)pLysS TB None 6.24 0.33 7.22 6.21 Example 1 pVP65KR-gab-SacB(-) XL10-Gold TB None 7.23 0.53 7.84 7.55

<110> SEJONG UNIVERSITY INDUSTRY ACADEMY COOPERATION FOUNDATION <120> Auto-induction Plasmid <130> 20P02019 <150> KR 10-2019-0055078 <151> 2019-05-10 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> gab promoter sequence <400> 1 gccttgcttc cattgcggat gtaaaagcgg ctagtattta 40 <210> 2 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> forward primer sequence replacing with gab promoter <400> 2 ctatcactga tagggactcg agaaagcctt gcttccattg cggatgtaaa agcggctagt 60 atttatttca cacagaattc attaaag 87 <210> 3 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> reverse primer sequence replacing with gab promoter <400> 3 ctttaatgaa ttctgtgtga aataaatact agccgctttt acatccgcaa tggaagcaag 60 gctttctcga gtccctatca gtgatag 87 <210> 4 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer sequence amplifying levansucrase gene <400> 4 ggttgcgatc gccatgaaca tcaaaaagtt tgcaaaacaa gc 42 <210> 5 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> primer sequence amplifying levansucrase gene <400> 5 ccttgaacaa ggacaattaa cagttaacaa attgtttaaa ctgaattcga gctcacac 58 <210> 6 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer sequence removing HindIII site <400> 6 atgttcagca ggaagctcgg cgcaaacgtt gattg 35 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer sequence removing HindIII site <400> 7 caatcaacgt ttgcgccgag cttcctgctg aacat 35 <210> 8 <211> 7174 <212> DNA <213> Artificial Sequence <220> <223> pVP65K vector sequence <400> 8 cgtacgtcta gggcggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga 60 taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctctagcacg 120 cgtatcattg gatctttatt agtccatgat ggcggcaaca gtttttttgg ccatgaagtc 180 atcttccggc gcatcttcaa ccagggtaaa ggaaccccag ctgattttat ccgcgttgaa 240 tttccagttt ttgcaccaac catccgcggc atcgagataa gtcgccacga atttttccgg 300 aaattccacg aagtagttgc taccgttggt ggtatgagtc aggctgtgga tgcccagaat 360 gttgccatca atgatggaaa ctagtgggct gccacactgg ccatctttag tggtgatcca 420 gtgctgccag aaagaagtgt cttctttatg cacaatgtga gaggattcag acaccaggct 480 ggacacgctt ttctgctgaa agttggtgga caccatgcac acacgatctt tgatggtcgg 540 ctgacggaat ttcagtttct gcgggaacgg cgggaagtct ttagccattt tgataacgat 600 aatgtcacgg ccttcaaccg gtttcatctg cagctgggta gagtttttga ctttgaattc 660 accgtgcatg gttttgatgg tcagttcgcc attgttacga cgaaacagat gctggttggc 720 aatgatatac gggccaaaac caatgccaaa cagacgttca ctatgaccat ccgaggagtt 780 ttccagcagg catacgcaag cagagatcgg attaaaatcg cgcacgccct tcagcaaagc 840 cttagacata tcttacctcc ttaaatcaat tcggtcagtg cgtcctgctg atgtgctcag 900 tatctctatc actgataggg atgtcaatct ctatcactga tagggactcg agaaatcata 960 aaaaatttat ttgctttgtg agcggataac aattataata gattcaattg tgagcggata 1020 acaatttcac acagaattca ttaaagagga gaaattaacc atgggtaaaa tcgaagaagg 1080 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa 1140 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga 1200 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga 1260 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt 1320 ccaggacaag ctgtatccgt ttacctggga tgcggtacgt tacaacggca agctgattgc 1380 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc 1440 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag 1500 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg 1560 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa 1620 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa 1680 tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac 1740 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac 1800 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg 1860 tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct 1920 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa 1980 gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccactatgg aaaacgccca 2040 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac 2100 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca 2160 gactttaatt aacggcgacg gtgccgggga aaccgtgcgt ttccagtctc atcaccatca 2220 tcaccatcac catgcatccg cgatcgcggc cgcgttggaa taagtaaagg aatcacatgg 2280 cacaggttat caacacgttt gacggggttg cggattatct tcagacatat cataagctac 2340 ctgataatta cattacaaaa tcagaagcac aagccctcgg ctgggtggca tcaaaaggga 2400 accttgcaga cgtcgctccg gggaaaagca tcggcggaga catcttctca aacaaggaag 2460 gcaaactccc gggcaaaagc ggacgaacat ggcgtgaagc ggatattaac tatacatcag 2520 gcttcagaaa ttcagaccgg attctttact caagcgactg gctgatttac aaaacaacgg 2580 accattatca gacctttaca aaaatcagat aattaggcac cccaggcttt acactttatg 2640 ctttcggctc gtataatgtg tggattttga gttaggatcc gtcgagattt tcaggagcta 2700 aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc 2760 atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg 2820 ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc 2880 cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa 2940 tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg 3000 agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc 3060 tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag 3120 ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg 3180 atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcacgatg ggcaaatatt 3240 atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg 3300 atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg 3360 gcggggcgta atgtttaaac gaattcgagc tcggtacccg gggatcctct agagtcgacc 3420 tgcaggcatg caagctgatc cggctgctaa caaagcccga aaggaagctg agttggctgc 3480 tgccaccgct gagcaataac tagcaaactc gtttctcgtt cagctttctt gtacaaagtg 3540 gtgatggcgc gcctgtagga cgtcgacggt accatcgata cgcgttcgaa gcttcgcctg 3600 gggtaatgac tctctagctt gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg 3660 gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccc 3720 tctagattac gtgcagtcga tgataagctg tcaaacatga gaattgtgcc taatgagtga 3780 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 3840 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc 3900 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg 3960 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt 4020 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact 4080 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc 4140 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc 4200 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga 4260 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa 4320 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg 4380 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag 4440 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg 4500 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc 4560 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc 4620 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga 4680 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg 4740 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa 4800 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct 4860 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg 4920 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtcgga acctagagct 4980 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg 5040 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg 5100 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata 5160 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga 5220 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct 5280 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 5340 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 5400 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 5460 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 5520 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 5580 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 5640 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 5700 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 5760 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 5820 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 5880 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 5940 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 6000 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 6060 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 6120 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 6180 atatgagtaa acttggtctg acagccctag gtcagaagaa ctcgtcaaga aggcgataga 6240 aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc 6300 attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt 6360 ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga 6420 tattcggcaa gcaggcatcg ccatgagtca cgacgagatc ctcaccatcc ggcatacgcg 6480 ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat 6540 cctgatcgac aagaccggct tccatccgag tgcgtgctcg ctcgatgcga tgtttcgctt 6600 ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca 6660 tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt 6720 cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag 6780 gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg 6840 caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca 6900 cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca 6960 cccaagcggc cggagaacct gcgtgcaatc catcttgttc aagcatgcga aacgaccgtc 7020 atcctgtctc ttgatcagat cttgatcccc tgcgccatca gatccttggc ggcaagaaag 7080 ccatccagtt tactttgcag ggcttcccaa ccttaccaga gggcgcccca gctggcaatt 7140 cttttgaagc tcacgctgcc gcaagcactc aggg 7174 <210> 9 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> T5 promoter sequence in pVP65K vector <400> 9 tcataaaaaa tttatttgct ttgtgagcgg ataacaatta taata 45 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> lac operator sequence in pVP65K vector <400> 10 ttgtgagcgg ataacaa 17 <210> 11 <211> 2000 <212> DNA <213> Artificial Sequence <220> <223> MBP-mCherry-8xHis gene block <400> 11 gaaattaacc atgggtaaaa tcgaagaagg taaactggta atctggatta acggcgataa 60 aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt 120 caccgttgag catccggata aactggaaga gaaattccca caggttgcgg caactggcga 180 tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct 240 gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga 300 tgcggtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct 360 gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct 420 ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta 480 cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa 540 gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct 600 ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc 660 tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat 720 cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc 780 caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct 840 ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa 900 agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc 960 acgtattgcc gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca 1020 gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca 1080 gactgtcgat gaagccctga aagacgcgca gactatggtt tccaagggcg aggaggataa 1140 catggctatc attaaagagt tcatgcgctt caaagttcac atggagggtt ctgttaacgg 1200 tcacgagttc gagatcgaag gcgaaggcga gggccgtccg tatgaaggca cccagaccgc 1260 caaactgaaa gtgactaaag gcggcccgct gccttttgcg tgggacatcc tgagcccgca 1320 atttatgtac ggttctaaag cgtatgttaa acacccagcg gatatcccgg actatctgaa 1380 gctgtctttt ccggaaggtt tcaagtggga acgcgtaatg aattttgaag atggtggtgt 1440 cgtgaccgtc actcaggact cctccctgca ggatggcgag ttcatctata aagttaaact 1500 gcgtggtact aattttccat ctgatggccc ggtgatgcag aaaaagacga tgggttggga 1560 ggcgtctagc gaacgcatgt atccggaaga tggtgcgctg aaaggcgaaa ttaaacagcg 1620 cctgaaactg aaagatggcg gccattatga cgctgaagtg aaaaccacgt acaaagccaa 1680 gaaacctgtg cagctgcctg gcgcgtacaa tgtgaatatt aaactggaca tcacctctca 1740 taatgaagat tatacgatcg tagagcaata tgagcgcgcg gagggtcgtc attctaccgg 1800 tggcatggat gagctgtaca aaggggaaac cgtgcgtttc cagtctcatc accatcatca 1860 ccatcaccat gcatccgcga tcgcggccgc gttggaataa gtaaaggaat cacatggcac 1920 aggttatcaa cacgtttgac ggggttgcgg attatcttca gacatatcat aagctacctg 1980 ataattacat tacaaaatca 2000 <210> 12 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> barnase sequence in pVP65KR vector <400> 12 atggcacagg ttatcaacac gtttgacggg gttgcggatt atcttcagac atatcataag 60 ctacctgata attacattac aaaatcagaa gcacaagccc tcggctgggt ggcatcaaaa 120 gggaaccttg cagacgtcgc tccggggaaa agcatcggcg gagacatctt ctcaaacaag 180 gaaggcaaac tcccgggcaa aagcggacga acatggcgtg aagcggatat taactataca 240 tcaggcttca gaaattcaga ccggattctt tactcaagcg actggctgat ttacaaaaca 300 acggaccatt atcagacctt tacaaaaatc agataa 336 <210> 13 <211> 1421 <212> DNA <213> Artificial Sequence <220> <223> Levansucrase (SacB) sequence in pVP65KR vector <400> 13 atgaacatca aaaagtttgc aaaacaagca acagtattaa cctttactac cgcactgctg 60 gcaggaggcg caactcaagc gtttgcgaaa gaaacgaacc aaaagccata taaggaaaca 120 tacggcattt cccatattac acgccatgat atgctgcaaa tccctgaaca gcaaaaaaat 180 gaaaaatatc aagttcctga attcgattcg tccacaatta aaaatatctc ttctgcaaaa 240 ggcctggacg tttgggacag ctggccatta caaaacgctg acggcactgt cgcaaactat 300 cacggctacc acatcgtctt tgcattagcc ggagatccta aaaatgcgga tgacacatcg 360 atttacatgt tctatcaaaa agtcggcgaa acttctattg acagctggaa aaacgctggc 420 cgcgtcttta aagacagcga caaattcgat gcaaatgatt ctatcctaaa agaccaaaca 480 caagaatggt caggttcagc cacatttaca tctgacggaa aaatccgttt attctacact 540 gatttctccg gtaaacatta cggcaaacaa acactgacaa ctgcacaagt taacgtatca 600 gcatcagaca gctctttgaa catcaacggt gtagaggatt ataaatcaat ctttgacggt 660 gacggaaaaa cgtatcaaaa tgtacagcag ttcatcgatg aaggcaacta cagctcaggc 720 gacaaccata cgctgagaga tcctcactac gtagaagata aaggccacaa atacttagta 780 tttgaagcaa acactggaac tgaagatggc taccaaggcg aagaatcttt atttaacaaa 840 gcatactatg gcaaaagcac atcattcttc cgtcaagaaa gtcaaaaact tctgcaaagc 900 gataaaaaac gcacggctga gttagcaaac ggcgctctcg gtatgattga gctaaacgat 960 gattacacac tgaaaaaagt gatgaaaccg ctgattgcat ctaacacagt aacagatgaa 1020 attgaacgcg cgaacgtctt taaaatgaac ggcaaatggt acctgttcac tgactcccgc 1080 ggatcaaaaa tgacgattga cggcattacg tctaacgata tttacatgct tggttatgtt 1140 tctaattctt taactggccc atacaagccg ctgaacaaaa ctggccttgt gttaaaaatg 1200 gatcttgatc ctaacgatgt aacctttact tactcacact tcgctgtacc tcaagcgaaa 1260 ggaaacaatg tcgtgattac aagctatatg acaaacagag gattctacgc agacaaacaa 1320 tcaacgtttg cgccaagctt cctgctgaac atcaaaggca agaaaacatc tgttgtcaaa 1380 gacagcatcc ttgaacaagg acaattaaca gttaacaaat t 1421

Claims (11)

Recombinant plasmid containing a promoter gene consisting of the sequence of SEQ ID NO: 1.
The plasmid of claim 1, wherein the gene encoding the protein of interest is operatively linked with the promoter.
The plasmid of claim 1, wherein the plasmid has a promoter gene having an operator replaced with a promoter gene consisting of the sequence of SEQ ID NO: 1.
Host microorganism transformed with a recombinant plasmid containing a promoter gene consisting of the sequence of SEQ ID NO: 1.
The microorganism according to claim 4, wherein the gene encoding the protein of interest is operatively linked with the promoter.
The microorganism according to claim 4, wherein the plasmid has a promoter gene having an operator substituted with a promoter gene consisting of the sequence of SEQ ID NO: 1.
The microorganism of claim 4, wherein the microorganism is at least one strain selected from the group consisting of E. coli, Pseudomonas, cyanobacteria, and Bacillus.
A composition for producing a protein comprising the plasmid of any one of claims 1 to 3 or the microorganism of any one of claims 4 to 7.
A method for producing a protein of interest comprising culturing the microorganism of any one of claims 4 to 7.
The method of claim 9, further comprising recovering the protein of interest.
The method of claim 9, wherein the culture is performed in a TB medium.

Images