WO2010064861A2 - Variante de promoteurs et procédé pour produire une protéine à l'aide de celle-ci - Google Patents

Variante de promoteurs et procédé pour produire une protéine à l'aide de celle-ci Download PDF

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WO2010064861A2
WO2010064861A2 PCT/KR2009/007209 KR2009007209W WO2010064861A2 WO 2010064861 A2 WO2010064861 A2 WO 2010064861A2 KR 2009007209 W KR2009007209 W KR 2009007209W WO 2010064861 A2 WO2010064861 A2 WO 2010064861A2
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bacillus
promoter
vector
host cell
protein
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WO2010064861A3 (fr
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최수근
반재구
박승환
이수진
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한국생명공학연구원
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a Bacillus thringiensis cry3Aa promoter variant, wherein any one or more of -35, space, and -10 sites of the Bacillus thuringiensis cry3Aa promoter are mutated downstream.
  • Bacillus has been used with Escherichia coli and yeast as a major host to produce industrially useful proteins.
  • the advantages of Bacillus as a protein-producing host are: (1) harmless to the human body, (2) capable of secreting large amounts of proteins extracellularly, (3) producing endotoxins, and (4) GRAS (generally) by the FDA. regared as safe, (5) free from codon bias, (6) easy to manipulate, transform, and mass culture, and (7) genomic information is known (Shumann W. 2007. Adv. Appl. Microbiol. 62: 137-189).
  • Currently, about 60% of the world's industrial enzymes are produced by Bacillus as a host bacterium (Westers L. 2004.
  • Biochimica et Biophysica Acta 1694: 299-310) due to the characteristics of GRAS (generally regarded as safe) microorganisms, which are harmless to humans. It is known to be suitable for the production of food or pharmaceutical proteins (Schallmey M. 2004. Can. J. Microbiol. 50: 1-17.).
  • Escherichia coli which is widely used as an expression host, requires physical, chemical or enzymatic cell destruction methods because most of the expressed proteins are present in cells (Witte A and Lubitz W. 1989. Eur. J. Biochem. 180: 393-398.). Therefore, inexpensive methods for cell destruction are indispensable for mass production of target proteins.
  • Bacillus cells are automatically destroyed until late in the phase of cell growth, and proteins in the cells are exposed to the medium (Nugroho FA et al., 1999. J. Bacteriol. 181: 6230-6237).
  • Bacillus thuringiensis is a Gram-positive bacillus that is characterized by producing toxin protein crystals in amounts of up to 25-30% of the cell dry weight when spores are formed.
  • the toxin protein crystals produced from Bacillus thuringiensis have a specific insecticidal effect against insects, and thus become a major active ingredient in the preparation of microbial insecticides.
  • about 450 species of toxin protein genes have been isolated and identified. http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/).
  • cry1A gene is sequentially transcribed after the logarithmic phase by two promoters, BtI and BtII, Brown and Whiteley. Reported that the two promoters are transcribed by ⁇ 35 and ⁇ 28 homologous to ⁇ E and ⁇ K of Bacillus thermos (Proc. Natl. Acad. Sci. USA, 85: 4166-4170 J. Bacteriol., 172: 6682-6688, 1988). Most of the toxin protein genes have similar promoter structures, but the cry3A gene in the toxin protein genes is an exceptional case.
  • the inventors of the present invention can develop a mass expression system of a target protein by improving cry3Aa, and an efficient system capable of stably mass-expressing an enzyme or a protein without affecting cell growth.
  • a promoter variant capable of significantly increasing the expression amount of a downstream gene was prepared, and the target protein from Gram-positive bacteria was measured by measuring the expression level of protease using the promoter variant. It was confirmed that the mass production can be completed the present invention.
  • An object of the present invention is to provide a polynucleotide having an increased gene expression downstream by mutation of any one or more of the -35 site, the space site and the -10 site of the Bacillus thuringiensis cry3Aa promoter. will be.
  • Another object of the present invention is to provide a vector comprising the polynucleotide sequence.
  • Still another object of the present invention is to provide a host cell comprising the vector.
  • Still another object of the present invention is to prepare a vector comprising a cry3Aa promoter variant; (b) introducing the prepared vector into a host cell; And (c) to provide a protein production method comprising the step of recovering the target protein from the host cell.
  • the expression system of the present invention it is possible to stably mass-express useful enzymes or proteins without affecting the growth of cells even without a specific expression inducing factor, and to efficiently recover the produced proteins, thereby being industrially useful. It can be effectively used for the production of proteins.
  • 1 is a comparative analysis of the expression levels of beta galactosidase using D32, cry1Aa and aprE promoters in Bacillus cilia.
  • FIG. 2 is a diagram showing the nucleotide sequence of the promoter D32 and variants D52, D82 and D92. The broken arrow indicates the transfer start position.
  • Figure 3 is a comparative analysis of the amount of beta galactosidase expression using promoter D32 and variants D52, D82, and D92 in Bacillus cilia.
  • Figure 4 is a comparison of the expression levels of the protease AprE using promoter D32 and variants D52, D82 and D92 in Bacillus 30. Bacillus cerilias 168 was used as a control.
  • FIG. 5 is a diagram showing the protein electrophoresis of the expression level of protease AprE using promoter D32 and variants D52, D82, and D92 in Bacillus sertilis. Bacillus cerilias 168 was used as a control. Arrows indicate expressed proteases.
  • the present invention provides a poly-modified poly (R) gene having an increased gene expression downstream by mutating any one or more of -35, space, and -10 sites of the Bacillus thringiensis cry3Aa promoter. It relates to nucleotides.
  • Bacillus thuringiensis of the present invention is a gram-positive bacterium belonging to the genus Bacillus. It penetrates into insects and produces toxins, and is also used as a source of pesticides. In the present invention, it is possible to induce mass production of proteins using the Bacillus thuringiensis promoter sequence.
  • the Bacillus thuringiensis promoter can be obtained from various subspecies of Bacillus thuringiensis, preferably B. thuringiensis sub. Alesti, B. thuringiensis sub. Sotto, and Morrisonia ( B. thuringiensis sub.morrisoni, B. thuringiensis sub.sandiego, B. thuringiensis sub.darmstadiensis, B.
  • the term "promoter” refers to a non-translated nucleic acid sequence upstream of a coding region that contains a binding site for polymerase and has a transcription initiation activity of a promoter downstream gene into mRNA.
  • the present invention can control the expression level of the downstream gene by manipulating the polynucleotide sequence of a specific promoter.
  • the present invention can provide a method for mass production of proteins by using a site having a transcription initiation activity of a Bacillus thuringiensis promoter that is specifically induced at cell growth arrest.
  • the promoter used in the present invention is cry3Aa of Bacillus thuringiensis, in a preferred embodiment of the present invention produced variants for specific regions of the cry3Aa promoter.
  • the promoter of the present invention contains a -35 site, a space site and a -10 site as binding sites for the polymerase.
  • the -35 site, the space site and the -10 site of the cry3Aa promoter used in the present invention indicate ttgcaa, ttgaagaattattaatgt and taagct in the nucleotide sequences of SEQ ID NO: 1, respectively, and in a preferred embodiment of the present invention, the nucleotide sequences of the sites Variants inducing mutations were prepared.
  • the -35 site is TTGACA
  • the space site is 17bp in size
  • the -10 site was prepared a mutated polynucleotide mutant, characterized in that the mutated to TATAAT, preferably of three sites Mutations can occur individually or in combination thereof.
  • the cry3Aa promoter of the present invention may further include a construct consisting of a polynucleotide sequence required for expression downstream of the promoter for efficient expression of a target protein.
  • the structure includes a leader and / or a stab.
  • “Reader” refers to a non-translated nucleic acid sequence upstream of the coding region of mRNA transcribed by a promoter.
  • the leader of the present invention is present between the Bacillus thuringiensis cry3Aa promoter and stab and may have a nucleotide sequence set forth in SEQ ID NO: 2.
  • stab refers to a sequence that contributes to stabilization of mRNA in a non-translated nucleic acid sequence upstream of the coding region of mRNA transcribed by a promoter, and preferably the stab of the present invention is a subordinate of a Bacillus thuringiensis cry3Aa leader. It may have a nucleotide sequence present in SEQ ID NO: 3 described in.
  • Promoter variants of the invention also include promoter nucleic acid sequences in which one or more nucleic acid bases have been modified by substitution, deletion, insertion, or combination thereof, so long as they retain promoter activity. By producing a promoter having increased activity compared to a natural promoter through such sequence variation, it is possible to improve the function of the promoter appropriately.
  • Such mutations can be made by a variety of methods known in the art, examples of which include error-prone PCR, DNA shuffling, site-directed mutations. mutagenesis) method.
  • the variant of the invention may have a nucleotide sequence set forth in SEQ ID NO: 4, 5, or 6 (named promoters D52, D82 and D92, respectively) and may include a leader or stab downstream thereof.
  • the variant may be prepared from a promoter sequence as set forth in SEQ ID NO: 1 (hereinafter referred to as "D32" promoter) using genetic recombination techniques and PCR methods.
  • the present invention comprises a variant set forth in SEQ ID NO: 4, 5 or 6, or a promoter sequence whose substantial activity is similar to that of the variant activity by variation of the D32 proboter.
  • "Homologous” refers to the same activity with a nucleic acid sequence of a variant of the wild type or the same activity, and homology comparison is homology between two or more sequences using a comparison program that is easy to see with the naked eye or to purchase Can be calculated as a percentage. It is apparent to those skilled in the art that homology with the promoter sequence is equivalent to the nucleotide sequence derived from the present invention as long as it retains the promoter activity for gene expression desired in the present invention.
  • This homologous sequence may be preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, of the nucleic acid sequence encoding the promoter region of SEQ ID NO: 4, 5, or 6 of the present invention, Most preferably at least 95% identical nucleic acid sequences.
  • the invention in another aspect, relates to a vector comprising said polynucleotide sequence.
  • vector refers to a gene construct, which is an expression vector capable of expressing a protein of interest in a suitable host cell, and which contains essential regulatory elements operably linked to express the gene insert.
  • Expression vectors associated with the present invention are vectors in which the promoter is a variant of cry3Aa promoter D32, including plasmid vectors (e.g., pSC101, ColE1, pBR322, pUC8 / 9, pHC79 and pUC19, etc.), cosmid vectors, bacteriophage vectors (e.g. ⁇ gt4). ? B,? -Charon, ?? z1 and M13), yeast vectors, viral vectors and the like.
  • Viral vectors are retroviruses, such as Human immunodeficiency virus HIV (Murine leukemia virus) MLV (Avian sarcoma leukosis virus), SNV (Spleen necrosis virus), RSV (Rous sarcoma virus), MMTV (Mouse mammary) tumor viruses), including but not limited to vectors derived from Adenovirus, Adeno-associated virus, Herpes simplex virus, and the like.
  • the vector was prepared by cutting the plasmid pDG1661 using EcoRI and BamHI and inserting the cry3Aa variant sequence.
  • operably linked refers to a functional link between a promoter variant nucleic acid sequence and a nucleotide sequence encoding a protein of interest to perform a general function.
  • Operative linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art and the like.
  • regulatory element refers to a non-translated nucleic acid sequence that assists or influences the enhancement of transcription, translation or expression of a nucleic acid sequence encoding a protein.
  • Expression vectors of the present invention essentially include a variant of the D32 promoter as a regulatory element, and expression control sequences that may affect the expression of proteins, e.g., start codons, stop codons, polyadenylation signals, enhancers, membranes Signal sequences for targeting or secretion, and the like.
  • Enhancer sequences are nucleic acid sequences that are located at various sites in the promoter and increase transcriptional activity as compared to the transcriptional activity by the promoter in the absence of the enhancer sequence.
  • the signal sequence includes PhoA signal sequence and OmpA signal sequence when the host is Escherichia spp., And ⁇ -amylase signal sequence and subtilisin signal sequence when the host is Bacillus.
  • MF ⁇ signal sequence, SUC2 signal sequence, etc. if the host is an animal cell, insulin signal sequence, ⁇ -interferon signal sequence, antibody molecule signal sequence, etc. may be used, but is not limited thereto.
  • the vector when the vector is a replicable expression vector, the vector may include a replication origin, which is a specific nucleic acid sequence from which replication is initiated.
  • the vector may include a selection marker.
  • the selection marker is for selecting cells transformed with the vector, and markers conferring a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins can be used. Since only cells expressing a selection marker survive in an environment treated with a selective agent, transformed cells can be selected.
  • Representative examples of the selection markers include ura4, leu1, his3, and the like, which are nutritional markers, but the types of selection markers that can be used in the present invention are not limited by the above examples.
  • the nucleic acid sequence encoding the target protein with the vector is inserted and expressed downstream of the promoter.
  • the target protein that can be inserted into the vector is not particularly limited, but medical and industrially useful target proteins include hormones, cytokines, enzymes, coagulation factors, transport proteins, receptors, regulatory proteins, structural proteins, transcription factors, antigens, antibodies, and the like. have.
  • a protease activity of the Bacillus strain including the vector is prepared by operably linking aprE, which is one of the protease enzymes of Bacillus thriller, downstream of the variant promoter sequence. Measured.
  • the present invention relates to a host cell comprising the vector.
  • the term "host cell” refers to a cell that provides nutrition by parasitic other microorganisms or genes, and refers to a cell that has various genetic or molecular effects in the host cell by transforming the vector into the host cell. do.
  • the host cell can be inserted with foreign DNA, such as a vector, in a competence state that can accept foreign DNA.
  • the host cell is provided with the genotype of the vector. .
  • the host cell of the present invention may be Gram positive uniform, for example Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus cereus ), Bacillus thuringiensis, Bacillus brevis, Bacillus brevis, Lactobacillus genus, and bacteria of the genus Paenibacillus, etc., but the vector of the present invention can be transformed by the above examples.
  • the host cell in which it is located is not limited.
  • a method of introducing a vector into the host cell may be used a transformation method.
  • Transformation refers to a phenomenon in which DNA is introduced into a host so that the DNA can be reproduced as a factor of a chromosome or by completion of chromosome integration, thereby introducing an external DNA into a cell and causing an artificial genetic change.
  • any transformation method may be used, and may be easily performed according to a conventional method in the art.
  • transformation methods include the CaCl 2 precipitation method, the CaCl 2 method, a Hanahan method that improves efficiency by using a reducing agent called DMSO (dimethyl sulfoxide), electroporation, calcium phosphate precipitation, plasma fusion method, silicon carbide Agitation with fibers, agro bacteria mediated transformation, transformation with PEG, dextran sulfate, lipofectamine and dry / inhibition mediated transformation.
  • DMSO dimethyl sulfoxide
  • electroporation calcium phosphate precipitation
  • plasma fusion method silicon carbide Agitation with fibers
  • agro bacteria mediated transformation transformation with PEG, dextran sulfate, lipofectamine and dry / inhibition mediated transformation.
  • the method for transforming pDZ-mqp of the present invention is not limited to the above examples, and transformation methods commonly used in the art may be used without limitation.
  • the present invention relates to a method of inducing expression of a gene of interest using the variant polynucleotide sequence of the cry3Aa promoter described above.
  • the vector containing the cry3Aa promoter sequence was named pD32, and the vector was used to compare the expression rate with pD52, pD82, and pD92, which are plasmid vectors containing variant sequences.
  • the present invention is prepared by inserting a protease into the plasmid vector, and then introduced into a Bacillus strain through a conventional transformation method used in the art, and cultured in a medium, thereby expressing a protein of interest as a protein of interest.
  • the Bacillus Stylus protease AprE (pD52-aprE, pD82-aprE, and pD92-aprE) is introduced to try to express, while transforming Bacillus Stylus 168, LB Production of AprE protein was confirmed by SDS-PAGE by culturing in the medium.
  • the variant nucleic acid molecule of the D32 promoter of the present invention can induce high expression of a gene of interest operably linked in a cell growth arrest phase.
  • the present inventors have used the PCR for cry3Aa promoter D32 (SEQ ID NO: 1) from the Bacillus thuringiensis isolate Bacteria BR31 (Park SH. Et al. 1997. Kor. J. Appl. Microbiol. Biotechnol. 25: 159-165) chromosome.
  • D32 promoter variants D52, D82, and D92 were prepared using genetic recombination technology and PCR using the D32 promoter.
  • the expression level of promoter variant D52 (SEQ ID NO: 4) was increased about four times compared to D32 (SEQ ID NO: 1), and D82 (SEQ ID NO: 5) and D92 (SEQ ID NO: 6) increased about 6 times the amount of expression compared to D32 (see Figure 3). That is, as a result of comparing and analyzing the expression amount of the protease using the variant, overexpression was not performed in the natural promoter (D32), but overexpression of the protease was observed in the variant promoters (D52, D82, and D92) (FIG. 5).
  • the present invention provides a method for preparing a vector comprising: (a) preparing a vector comprising a cry3Aa promoter variant; (b) introducing the prepared vector into a host cell; And (c) relates to a protein production method comprising the step of recovering the target protein from the host cell.
  • the cry3Aa promoter variant vector of the present invention can be prepared using restriction enzyme and PCR methods for vectors commonly used in the art as described above.
  • the prepared vector may be prepared by introducing into a host cell by a general transformation method, in a preferred embodiment of the present invention was introduced into Bacillus sertilis 168 using a natural introduction method, the protein expression amount was measured.
  • the host cell transformed by the above method may be cultured through a culture method commonly used in the art as needed.
  • the present invention was incubated for 16 hours in transformed Bacillus strains in LB medium to induce the production of the desired protein.
  • the present invention may further comprise the step of recovering the target protein in order to measure the amount of the target protein produced through the culture.
  • the cultured supernatant in order to compare the expression amount of AprE, the target protein, the cultured supernatant is mixed with azocaine dissolved in a PBS solution, reacted with TCA, and the culture supernatant is separated by centrifugation, followed by NaOH. The amount of protein expression was compared by mixing and measuring the absorbance.
  • sequences may be further included as needed in the preparation of the plasmid vector.
  • the additionally included sequence may be a tag sequence for protein purification, such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine Quiagen, USA), and most preferably 6x His (hexa histidine), but the examples do not limit the type of sequence required for purification of the target protein.
  • a fusion protein expressed by a vector containing the fusion sequence it may be purified by affinity chromatography.
  • glutathione-S-transferase when glutathione-S-transferase is fused, glutathione, which is a substrate of this enzyme, can be used, and when 6x His is used, a Ni-NTA His-binding resin column (Novagen, USA) can be used for desired protein. Can be easily recovered.
  • DNA containing the native cry3Aa promoter was prepared using primers cry3Aa-F3 (SEQ ID NO: 7), cry3Aa-R2 (SEQ ID NO: 13), and stab using stab-F (SEQ ID NO: 14) and stab-R2 (SEQ ID NO: 8), respectively.
  • PCR was obtained from the chromosome of Bacillus thuringiensis isolate bacteria BR31.
  • DNA containing the obtained cry3Aa promoter was digested with EcoRI and SpeI, and the stab was digested with SpeI and BamHI, and then inserted into the EcoRI and BamHI sites of plasmid pDG1661 to complete plasmid pD32.
  • cry1Aa promoter was expressed using Bacillus thuringiensis Custarki HD1 (Bacillus Genetic Stock Center, Ohio, USA) using cry1Aa-F (SEQ ID NO: 9) and cry1Aa-R (SEQ ID NO: 10). Obtained from the chromosome using PCR and the aprE promoter was Bacillus sertilis 168 (Kunst F. et al. 1997. Nature) using aprE-F (SEQ ID NO: 11) and aprE-R (SEQ ID NO: 12). 390: 249-256) was obtained using PCR.
  • the PCR products were digested with EcoRI and BamHI and cloned into plasmid pDG1661 and named pD1Aa-lacZ and pDaprE-lacZ, respectively.
  • the constructed plasmids pD32, pD1Aa-lacZ and pDaprE-lacZ were introduced into Bacillus sutilis 168 by natural introduction method, and pDaprE-lacZ was introduced from Bacillus certilis 1A165 (Bacillus Genetic Stock Center, Ohio, USA), in which the degU gene was mutated. Acquisition).
  • Bacillus certilis including plasmid pD32, pD1Aa-lacZ or pDaprE-lacZ, were inoculated in DSM medium (Harwood CR and Cutting SM. 1990. Molecular Biological Methods for Bacillus. John Wiley & Sons Ltd.) and cultured cells. Beta galactosidase activity was measured at 1 hour intervals. As a result, the strain containing pD32 showed low enzymatic activity in the cell growth phase but overexpression was induced in the growth arrester, and the expression level was about 30 times higher than that of cry1Aa (FIG. 1).
  • the aprE promoter which is widely used in industry, is known to significantly increase expression in degU32 mutant environments (Jan J. et al. 2001. Appl. Microbiol. Biotecnol. 55: 69-75). type) or 150-fold and 10-fold higher expression levels than the aprE promoter in the degU32 environment, respectively.
  • the aprE gene was obtained by PCR from the chromosome of Bacillus sertilis 168 using primers aprE-F1 (SEQ ID NO: 18) and aprE-R1 (SEQ ID NO: 19). The PCR products were digested with BamHI and BsiWI and cloned into the same site of plasmid pD32 and named pD32-aprE. The prepared plasmid pD32-aprE was introduced into Bacillus sertilis 168 by natural introduction.
  • AprE expression was determined by incubating the Bacillus strain including the plasmid pD32-aprE for 16 hours in LB medium, and the culture supernatant was analyzed by SDS-PAGE. As a result, expression of AprE could not be observed (see FIG. 5). Thus, overexpression of the target protein required improvement of the D32 promoter.
  • the nucleotide sequence of -35 and -10 sites of the cry3Aa promoter was changed to be similar to the corresponding site of ⁇ A of Bacillus thyrus (FIG. 2).
  • Promoter D52 having the -35 site consensus sequence was obtained from the plasmid pD32 by PCR using primers cry3Aa-F5 (SEQ ID NO: 15) and stab-R2 (SEQ ID NO: 8).
  • the secured D52 promoter was digested with EcoRI and BamHI and inserted into the EcoRI and BamHI sites of plasmid pDG1661 to complete plasmid pD52.
  • Promoter D82 having both -35 and -10 consensus sequences was obtained by PCR from plasmid pD32 using primers cry3Aa-F8 (SEQ ID NO: 16) and stab-R2 (SEQ ID NO: 8).
  • the secured D82 promoter was digested with EcoRI and BamHI and inserted into the EcoRI and BamHI sites of plasmid pDG1661 to complete plasmid pD82. Meanwhile, the space region between the -35 and -10 sites of the cry3Aa promoter is composed of 18 bases, but the base is consensus -35 site, -10 site and Promoter D92 was constructed to resemble the space region of 17 bases.
  • Promoter D92 was obtained by PCR from plasmid pD32 using primers cry3Aa-F9 (SEQ ID NO: 17) and stab-R2 (SEQ ID NO: 8). The obtained D92 promoter was digested with EcoRI and BamHI and inserted into the EcoRI and BamHI sites of plasmid pDG1661 to complete plasmid pD92. The completed plasmids pD52, pD82 and pD92 were introduced into the Bacillus certilis 168 strain by natural introduction.
  • promoter variant D52 showed an expression increase of about 4 times compared to D32, and promoter variants D82 and D92 increased expression amount by about 6 times compared to D32 (FIG. 3).
  • aprE gene was obtained by PCR from the chromosome of Bacillus sertilis 168 using primers aprE-F1 (SEQ ID NO: 18) and aprE-R1 (SEQ ID NO: 19). The PCR products were digested with BamHI and BsiWI and cloned into the same sites of plasmids pD52, pD82 and pD92 respectively and named pD52-aprE, pD82-aprE and pD92-aprE, respectively.
  • the prepared plasmids were introduced into Bacillus certilis 168 by natural introduction method. Bacillus strains containing the plasmids were incubated for 16 hours in LB medium, and the activity of protease was measured in the culture supernatant. Protease activity was measured by azocaine (azo-casein, Sigma-Aldrich) dissolved in PBS solution (NaCl 8g, KCl 0.2g, Na2HPO4 1.44g, KH2PO4 0.24g / L, pH7.4) at a concentration of 2mg / ml. 300 ⁇ l and 50 ⁇ l of the culture supernatant were mixed and reacted at 37 ° C.
  • azocaine azo-casein, Sigma-Aldrich

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  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne une variante de promoteurs de Bacillus thuringiensis cry3Aa, et en particulier un polynucléotide présentant un taux d'expression amélioré de gènes en aval par la mutation d'au moins une région sélectionnée parmi 35 régions, une région d'intervalle, et une région -10 d'un promoteur du Bacillus thuringiensis cry3Aa. L'invention concerne également un vecteur comprenant la séquence du polynucléotide, une cellule hôte comprenant le vecteur, et un procédé de production de protéines comprenant les étapes consistant à: (a) préparer un vecteur comprenant une variante du promoteur cry3Aa, (b) introduire le vecteur préparé dans une cellule hôte, et (c) récupérer la protéine cible de la cellule hôte.
PCT/KR2009/007209 2008-12-03 2009-12-03 Variante de promoteurs et procédé pour produire une protéine à l'aide de celle-ci WO2010064861A2 (fr)

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KR1020080122155A KR20100063579A (ko) 2008-12-03 2008-12-03 프로모터 변이체 및 이를 이용한 단백질 생산방법
KR10-2008-0122155 2008-12-03

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KR101280503B1 (ko) * 2010-02-03 2013-07-02 한국생명공학연구원 프로모터 변이체 및 이를 이용한 단백질 생산방법
KR101641578B1 (ko) * 2014-01-08 2016-07-21 한국생명공학연구원 프로모터 변이체 및 이를 이용한 단백질 생산방법
KR20230091593A (ko) * 2021-12-16 2023-06-23 경북대학교 산학협력단 단백질 자동 발현 시스템

Citations (3)

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US20020165139A1 (en) * 2000-06-29 2002-11-07 University Of Maryland, College Park Construction of a structural variant of sublancin to facilitate its isolation and use in bioremediation of environmental contamination by gram-positive spore formers such as Bacillus anthrasis
US20050112603A1 (en) * 2003-11-21 2005-05-26 Industrial Technology Research Institute Method for purification, modification and immobilization of recombinant protein
WO2008140615A2 (fr) * 2006-12-21 2008-11-20 Novozymes, Inc. Séquences stabilisatrices d'arn messager modifié permettant l'expression de gènes dans des cellules bactériennes

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Publication number Priority date Publication date Assignee Title
US20020165139A1 (en) * 2000-06-29 2002-11-07 University Of Maryland, College Park Construction of a structural variant of sublancin to facilitate its isolation and use in bioremediation of environmental contamination by gram-positive spore formers such as Bacillus anthrasis
US20050112603A1 (en) * 2003-11-21 2005-05-26 Industrial Technology Research Institute Method for purification, modification and immobilization of recombinant protein
WO2008140615A2 (fr) * 2006-12-21 2008-11-20 Novozymes, Inc. Séquences stabilisatrices d'arn messager modifié permettant l'expression de gènes dans des cellules bactériennes

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Title
AGAISSE, H. ET AL.: 'Structural and Functional Analysis of the Promoter Region Involved in Full Expression of the cryIIIA toxin gene of Bacillus thuringiensis' MOL. MICROBIOL. vol. 13, no. 1, 1994, pages 97 - 107 *
BIEDENDIEK, R. ET AL.: 'Plasmid System for the Intracellular Production and Purification of Affinity-Tagged Proteins in Bacillus megaterium' BIOTECHNOL. BIOENG. vol. 96, no. 3, 15 February 2007, pages 525 - 537 *

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WO2010064861A3 (fr) 2010-09-10

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