KR101453437B1 - Novel nucleic acids for enhancing gene expressions and a method for protein production using the same - Google Patents
Novel nucleic acids for enhancing gene expressions and a method for protein production using the same Download PDFInfo
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Abstract
The present invention relates to a novel stab nucleic acid molecule that enhances gene expression. The stab nucleic acid molecule can be included between a promoter and a target gene, thereby increasing the expression rate of a downstream target gene. More particularly, the present invention is Bacillus Chuo ringen sheath (Bacillus thuringiensis ) cry3Ca A novel stab nucleic acid molecule upstream of the gene and a novel stab nucleic acid molecule linked to the 5 'terminal nucleic acid molecule of the cry3Ca structural gene at the 3' end of the nucleic acid molecule.
The present invention also relates to a vector comprising the stab nucleic acid molecule, a host cell comprising the vector, and a method for producing a target protein from the host cell.
Description
The present invention relates to a novel stab nucleic acid molecule that enhances gene expression. The stab nucleic acid molecule can be included between a promoter and a target gene, thereby increasing the expression rate of a downstream target gene. More particularly, the present invention is Bacillus Chuo ringen sheath (Bacillus thuringiensis ) cry3Ca A novel stab nucleic acid molecule upstream of the gene and a novel stab nucleic acid molecule linked to the 5 'terminal nucleic acid molecule of the cry3Ca structural gene at the 3' end of the nucleic acid molecule.
The present invention also relates to a vector comprising the stab nucleic acid molecule, a host cell comprising the vector, and a method for producing a target protein from the host cell.
The mRNA transcribed from a specific gene is degraded in the cell after protein production. The degree of expression of the specific protein is controlled by the ratio of synthesis and degradation of the mRNA encoding the mRNA. In the case of higher organisms, mRNA degradation occurs by exoribonuclease in the 5 'to 3' direction (Muhlrad D et al., 1994, Genes Dev. 8: 855-866) Is first cleaved by an endoribonuclease called RNase E and then cleaved into small fragments of 2-5 nucleotides by RNase II, RNase R, PNPase and other 3 'to 5' And re-oligomerized and degraded to monomers by oligoribonuclease (Deutscher MP, 2006. Nucleic Acids Res. 34: 659-666, Ghosh S and Deutscher MP 1999. Proc. Natl. Acad. 96: 4372-4377).
In the case of microorganisms, an exoribonuclease that cleaves in the 5 'to 3' direction has not been found, so that the mechanism by which mRNA is degraded in E. coli has been considered to be general. However, when comparing the entire genome sequence of Gram-positive bacteria, Bacillus subtilis, with E. coli, two essential enzymes, Bacillus subtilis, RNase E and oligo ribonuclease were not found (Condon C and Putzer H. 2002. Nucleic Acids Res. 30: 5339-5346.). This means that the mRNA degradation mechanisms of Escherichia coli and Bacillus subtilis are different from each other. Recently, a new exoribonuclease RNase J1 has been found in Bacillus subtilis, which cleaves RNA in the 5 'to 3' direction (Mathy N et al. 2007. Cell 129: 681-692). Other microorganisms (Even S. 2005. Nucleic Acids Res. 33: 2141-2152). Therefore, mRNA degradation by RNase J1 is common in Gram-positive bacteria including Bacillus.
In addition, a bacterium having the activity of an exoribonuclease that cleaves RNA in the 5 'to 3' direction is a bacterium belonging to Archaea bacteria such as Halobacteriales , Methanobacteriales , Methanocortis Methanococcales , Methanopyrales , Methanosarcinales , Thermococcales and Nanoarchaeota. Gram-positive bacteria include Actinobacteria, Actinobacteria, (Firmicutes) alpha proteobacteria (alpha-proteobacteria) that the Gram methyl-negative bacteria, and the like including in tumefaciens (Methylobacterium) genus, Rhizopus emptying (Rhizobium) genus, and Agrobacterium (Agrobacterium), ring Nella ( Wolinella) genus Helicobacter (Helicobacter) and the genus Campylobacter (epsilon proteobacteria (epsilon-proteobacteria) and cyanobacteria (C including the genus Campylobacter) (Nucleic Acids Res. 2005. 33: 2141-2152).
In Bacillus strains, there are very stable mRNAs, which have a secondary structure at the 5 'end, or bind specific proteins or ribosomes to protect the mRNA (Agaisse H and Lereclus D. 1996. Mol. Microbiol. 20: USA 84: 498-502., Glatz E et al 1996. Mol. Microbiol. 19: 319-328., Hambraeus G. < RTI ID = 0.0 > , 2002: Microbiol 148: 1795-1803) .The protection of these 5 'ends hinders the degradation of mRNA by RNase J1, which helps to stabilize mRNA and ultimately increases the expression level of the sub-genes. In the case of the cry3Aa gene derived from Bacillus thuringiensis , there are two SD Shine-Dalgarno sequences above the structural gene of the transcribed mRNA (Agaisse H and Lereclus D. 1996. Mol. Microbiol. 20: 633- 643). The 30S ribosomes bind to the higher SD (STAB-SD), and the lower mRNA is degraded from RNase J1 , The 30S and 50S ribosomes bind to the lower SD sequence to detoxify the transcript between the promoter and the structural gene, which is due to the presence of the stab, the nucleotide sequence that stabilizes the transcript (Mathy N et al., 2007. Cell 129: 681-692) .The STAB-SD of cry3Aa has been reported to increase the expression level of a sub-gene even in combination with other promoters (Park HW et al., 1998. Appl. Microbiol. 64: 3932-3938). However, no systematic studies or studies on STAB-SD other than cry3Aa STAB-SD have yet been conducted.
Therefore, the inventors of the present invention have found a novel stab nucleic acid molecule upstream of the cry3Ca gene of Bacillus thuringiensis as a result of intensive efforts to find a sequence having other STAB-SD activity in addition to the above cry3Aa STAB-SD Furthermore, a longer nucleic acid molecule having the 5 'end of the cry3Ca gene bound to the 3' end of the nucleic acid molecule can also function as a novel stab nucleic acid molecule. Even when these novel stab nucleic acid molecules are positioned after other known promoters And that the expression level of downstream genes can be increased, thus completing the present invention.
An object of the present invention is Bacillus Chuo ringen sheath (Bacillus thuringiensis ) cry3Ca As a novel stab nucleic acid molecule having gene-based STAB-SD activity, it is intended to provide a novel stab nucleic acid molecule that increases the expression amount of a gene located downstream.
Another object of the present invention is to provide a vector comprising said stab nucleic acid molecule.
It is still another object of the present invention to provide a host cell comprising the vector.
Yet another object of the present invention is to provide a method for producing a vector comprising: (a) preparing a vector comprising a promoter, a stab nucleic acid molecule and a target gene; (b) introducing the vector into a host cell; And (c) recovering the target protein from the host cell.
In one aspect, the present invention relates to a novel stab nucleic acid molecule having a novel STAB-SD activity having the nucleotide sequence of SEQ ID NO: 1.
In another aspect, the present invention provides a nucleic acid molecule comprising: i) a nucleic acid molecule of SEQ ID NO: 1; and ii) a nucleic acid molecule at the 5'end of a cry3Ca structural gene linked to the 3'end of the nucleic acid molecule. Lt; RTI ID = 0.0 > stab < / RTI > nucleic acid molecule.
The term "STAB-SD" of the present invention refers to a site where 16S rRNA, which is a component of the 30S ribosome, selectively binds but is located at a distance of 100 bases or more from the start codon of the structural gene unlike the SD (Shine Dalgarno) As a site, "STAB-SD activity" refers to the activity of protecting RNA from exoribonuclease, and the activity of protecting Bacillus thuringiensis from RNase J1 And thus it is an activity that can increase the biline accumulation of the lower gene even when it binds to other promoter.
The term "stab nucleic acid molecule" of the present invention refers to a sequence that contributes to the stabilization of mRNA, which has STAB-SD activity among the toxic nucleic acid sequence upstream of the coding region of the mRNA transcribed by the promoter.
Preferably stab nucleic acid molecules of the present invention cry3Ca of Bacillus Chuo ringen sheath (Bacillus thuringiensis) A novel stab nucleic acid molecule having STAB-SD activity, which is identified between the promoter of the gene and the target gene and has the nucleotide sequence of SEQ ID NO: 1.
Preferably, the stab nucleic acid molecule of the present invention may be a stab nucleic acid molecule in which the nucleic acid molecule at the 5 'end of the cry3Ca structural gene is fused to the 3' end of the nucleic acid molecule of SEQ ID NO: 1.
In the present invention, the term "5 ' end of the structural gene" means from the translation start point of the gene, which means the N-terminal coding region of the structural gene. The N-terminus can be included without limitation to a portion capable of exhibiting STAB-SD activity.
More preferably, the stab nucleic acid molecule of the present invention may be the nucleotide sequence shown in SEQ ID NO: 2.
For the purposes of the present invention, those skilled in the art will appreciate that one or more nucleic acid bases of the stab nucleic acid molecule are mutated by substitution, deletion, insertion, or a combination thereof, as long as they retain the same STAB-SD activity. Therefore, preferably, the present invention can include all the sequences having homology to SEQ ID NO: 1 or 2, preferably 70% of the sequence, if the sequence can increase the expression of the downstream target gene Homology, more preferably 80%, even more preferably 90%, most preferably 95% or more homology.
According to a preferred embodiment of the invention, if a rather long stab nucleic acid molecules containing the portion 5 'end of cry3Ca structural gene derived from cry3Ca sikyeoteul in the rear phoB promoters to confirm that (Green Fluorescent Protein) GFP expression increased significantly ( 6), it was confirmed that the 5 'terminal of the structural gene of the corresponding gene derived from the stab nucleic acid molecule plays an important role in enhancing the gene expression of the stab nucleic acid molecule.
In the present invention, securing the stab nucleic acid molecule can be separated or prepared using standard molecular biology techniques. For example, using appropriate primer sequences. It may also be prepared using standard synthetic techniques using automated DNA synthesizers. In one specific embodiment of the present invention, cry3Ca For stab nucleic acid molecules, they were prepared by PCR using appropriate primer sequences.
Preferably, the stab nucleic acid molecule of the present invention exists between the promoter and the target gene, and can increase the expression of the target gene present downstream. This is because the novel stab nucleic acid molecule including STAB-SD prevents the 5'-terminal of RNA from being degraded by exoribonuclease and stabilizes the transcript through stabilization of the RNA structure. The 30S and 50S ribosomes bind to the base sequence, resulting in detoxification, thereby increasing the expression of the target gene.
The term "target gene " of the present invention means a downstream gene coding for a protein whose expression rate is desired to be increased, and any desired gene commonly used in the art can be used without limitation. Examples of the target protein useful for medicine and industrially include a hormone, a hormone analogue, an enzyme, an enzyme inhibitor, a fragment of a receptor and a receptor, an antibody and an antibody fragment, a monoclonal antibody, a structural protein, a toxin protein, By way of example, the example of a target gene that can be present downstream of the stab nucleic acid molecule of the present invention is not limited.
In another aspect, the present invention relates to a vector comprising the novel stab nucleic acid molecule. As used herein, the term "vector" refers to an expression vector capable of expressing a desired protein in a suitable host cell, including a necessary regulatory element operably linked to the expression of the gene insert. Expression vectors related to the present invention can be obtained by using plasmid vectors (e.g., pSC101, ColE1, pBR322, pUC8 / 9, pHC79 and pUC19 etc.), cosmid vectors, bacteriophage vectors (such as λgt4, λB, λ-Charon, λΔz1 and M13 ), Yeast vectors, viral vectors, and the like. Virus vectors include retroviruses such as HIV (human immunodeficiency virus), Murine leukemia virus (MLV), Avian sarcoma / leukosis (ASLV), Spleen necrosis virus (SNV), Rous sarcoma virus but are not limited to, vectors derived from tumor viruses, adenoviruses, adeno-associated viruses, herpes simplex viruses and the like. In a preferred embodiment of the present invention, plasmid pD32 (Korean Patent Application No. 10-2008-0122155), pAD-pSA4 (Korean Patent Application No. 10-2008-0074253), pDG-PSA4 (Korean Patent Application No. 10-2008-0074253 Was digested with SpeI and BamHI, and a vector was prepared by inserting the sequence of the obtained stab nucleic acid molecule.
In the present invention, "operably linked" refers to a functional linkage of a nucleic acid molecule sequence of a promoter, a stab nucleic acid molecule, and a nucleic acid molecule sequence encoding a desired protein to perform a general function. The operative linkage with the recombinant vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage are made using enzymes generally known in the art.
As used herein, the term " regulatory element "refers to a nucleic acid sequence that has been compromised to help or influence the transcription, translation or expression of a nucleic acid sequence encoding the protein. The expression vector of the present invention essentially comprises a stab nucleic acid molecule and may further include a promoter sequence commonly used in the art. As used herein, the term "promoter " refers to a toxin nucleic acid sequence that contains a binding site for a polymerase and has a transcription initiation activity to the mRNA of a gene located downstream. The promoter that can be located upstream of the stab nucleic acid molecule of the present invention can be any promoter used in the art, and is not limited thereto. Preferably, the promoter may be a promoter derived from a gram-positive bacterium. In a preferred embodiment of the present invention, the cry3Aa promoter of Bacillus thuringiensis or the promoter derived from Bacillus subtilis phoB By locating the promoter upstream of the stab nucleic acid molecule it was found that the expression of the target gene located downstream of the stab polynucleotide was increased (Figures 2, 4, and 6). In a preferred embodiment of the present invention, the cry3A [alpha] promoter and phoB The insertion of the promoter confirmed the increase in the expression of the downstream target gene due to the stab nucleic acid molecule (Examples 1 to 3). The expression vector of the present invention may also contain an expression control sequence capable of affecting the expression of the protein, for example, an initiation codon, a stop codon, a polyadenylation signal, an enhancer, a signal sequence for membrane targeting or secretion, etc. . The polyadenylation signal increases the stability of the transcript or facilitates cellular transport. The enhancer sequence is a nucleotide sequence that is located at various sites in the promoter and increases the transcriptional activity as compared with the transcriptional activity by the promoter in the absence of the enhancer sequence. The signal sequence includes the PhoA signal sequence and the OmpA signal sequence when the host is Escherichia genus, the α-amylase signal sequence and the subtilis signal sequence when the host is a genus of Bacillus sp. , An MF signal sequence, a SUC2 signal sequence, and the like. When the host is an animal cell, an insulin signal sequence, an a-interferon signal sequence, an antibody molecule signal sequence, and the like may be used.
In addition, if the vector is a replicable expression vector, it may contain a replication origin which is a specific nucleic acid sequence at which replication is initiated.
In addition, the vector may include a selection marker. Selection markers are for selecting cells transfected with a vector, and markers conferring selectable phenotypes such as drug resistance, nutritional requirement, resistance to cytotoxic agents or expression of surface proteins may be used. In the environment treated with the selective agent, only the cells expressing the selection marker survive, so that the transformed cells can be selected. Representative examples of selectable markers include auxotrophic markers ura4, leu1, his3, etc. However, the types of selectable markers usable in the present invention are not limited by the above examples.
The nucleic acid sequence encoding the target protein as the vector is inserted and expressed downstream of the promoter and the stab nucleic acid molecule. The target protein that can be inserted into the vector is not particularly limited, but examples of the target protein useful in medicine or industrially include hormones, cytokines, enzymes, coagulation factors, transport proteins, receptors, regulatory proteins, structural proteins, transcription factors, And the like. In a preferred embodiment of the present invention, a nucleotide sequence encoding β- galactosidase or GFP (Green Flourorescent Protein) was inserted to confirm the increase in the expression amount of the promoter by the stab
In yet another embodiment, the present invention relates to a host cell comprising the vector.
As used herein, the term "host cell" refers to a cell which parasitizes another microorganism or gene and supplies nutrition thereto. The term " host cell " means a cell in which a vector is transformed into a host cell to have various genetic or molecular effects do. Host cells can be injected with external DNA, such as a vector, in a competent state capable of accepting external DNA. When a vector is successfully introduced into a host cell, the host cell is provided with a genetic trait of the vector . The stab nucleic acid molecule of the present invention can stabilize RNA from exoribonuclease (exoribonuclease) and increase the efficiency of expression thereof. Thus, the host cell of the present invention is capable of cleaving RNA in the 5 'to 3' direction Any cell having the activity of exoribonucleic acidase can be used without limitation. Preferably, the host cell of the invention can be a gram-positive bacterium, an Archaea bacterium, or a gram-negative bacterium. Examples of gram positive bacteria include Bacillus subtilis subtilis), Bacillus Lee Kenny Po Ms (Bacillus licheniformis , Bacillus megaterium , Bacillus cereus , Bacillus thuringiensis or Brevibacillus < RTI ID = 0.0 > includes brevis), further Lactobacillus bacteria (Lactobacillus) genus Listeria (Listeria), A Companion Bacillus (Paenibacillus), An evil Martino My process (Actinomyces) genus Streptomyces (Streptomyces), A No-carboxylic Dia (Nocardia) genus Corynebacterium (Corynebacterium) in or Bifidobacterium (Bifidobacterium) but can include in, but not the type of gram-positive bacteria that can be used in the present invention by the example limits. Examples of Gram-negative bacteria, cyano bacteria (Cyanobacteria), preferably methyl tumefaciens (Methylobacterium) genus, Rhizopus emptying (Rhizobium) genus, Agrobacterium (Agrobacterium), A ring Nella (Wolinella) genus Helicobacter (Helicobacter) but not in or Campylobacter (Campylobacter) lay include bacteria, such as one, the type of Gram-negative bacteria with a vector of the present invention source by the transformant can be converted for example limited.
Preferably, a method of introducing a vector into a host cell may be a transformation method. "Transformation" refers to a phenomenon in which DNA is introduced into a host and DNA is replicable as a factor of chromosome or by completion of integration of chromosomes, introducing external DNA into a cell to cause artificial genetic change. Any transformation method can be used as the transformation method 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. The method for transforming the vector of the present invention is not limited to the above examples, and the transformation methods conventionally used in the art can be used without limitation.
In another aspect, the present invention relates to a method for inducing overexpression of a target gene using a specific promoter and the stab nucleic acid molecule.
When the stab nucleic acid molecule of the present invention is placed downstream of various promoters regardless of the type of the promoter of the gene from which the stab nucleic acid molecule is derived, overexpression of the target gene operably linked thereto can be induced, It is useful for proteins.
In a preferred embodiment of the present invention comprises a promoter sequence but cry3Aa cry3Aa were stab itself of the vector does not contain the nucleic acid molecule named pD3S0, Days (β- galactosidase) when beta-galactosidase promoter and get the desired gene using the vector A vector pD3CaS into which a stab nucleic acid molecule of stry nucleic acid molecule cry3Ca of the present invention (hereinafter, '3CaS1', SEQ ID NO: 1) was inserted between the genes was prepared (Fig. 1). In addition, the vector containing the Bacillus subtilis phoB promoter sequence was named pDG-PSA3, and the vector was used to insert the stab nucleic acid molecule 3CaS1 of the present invention between the promoter and the target gene, beta-galactosidase gene Vector pDG-P4CaS was prepared (Fig. 3). In addition, a vector pAD-pSA4 containing a Bacillus subtilis phoB promoter sequence was prepared, and a vector pAP16 in which the stab nucleic acid molecule of the present invention, 3CaS1, was inserted between the promoter of the vector and a GFP (Green Fluorescent Protein) , And a vector pAP17 in which a stab nucleic acid molecule (hereinafter referred to as' 3CaS2 'SEQ ID NO: 2) in which the 5' end of the cry3Ca structural gene was inserted was inserted into the 3CaS1 (FIG.
In the present invention, the plasmid vector is introduced into a strain of Bacillus genus through a conventional transformation method used in the art and cultured in a medium to confirm the expression amount of the target protein, beta-galactosidase. The 3CaS1 stab nucleic acid molecule increased the expression level of the cry3Aa promoter by about 10-fold (FIG. 2) as a result of comparing the expression level of beta-galactosidase used as the target protein.
In the case of the phoB promoter, 3CaS1 increased the expression level of beta-galactosidase about 1.7-fold (FIG. 4) and 3CaS2 increased the expression level of GFP about 13-fold (FIG. 6).
In another embodiment, the present invention provides a method of producing a vector comprising: (a) preparing a vector comprising a promoter, a stab nucleic acid molecule and a target gene; (b) introducing the produced vector into a host cell; And (c) recovering the target protein encoded by the target gene from the host cell.
The promoter of the present invention can be any promoter used in the art and can be inserted into the stab upstream of the vector using restriction enzymes and PCR methods. In addition, the prepared vector can be prepared by introducing the vector into a host cell by a general transformation method. In a preferred embodiment of the present invention, the vector is introduced into Bacillus subtilis 168 using a natural selection method and the amount of protein expression is measured.
In addition, the host cell transformed by the above-mentioned method may be cultured through a culture method conventionally used in the art if necessary. Preferably, the transformed Bacillus strain is cultured in DSM medium and CLPYG medium to induce production of the desired protein.
In order to facilitate the purification of the recovered target protein in the present invention, other sequences may be additionally included as needed in the production of the plasmid vector. The additional sequence may be a tag sequence for protein purification, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His ; Quiagen, USA), and most preferably 6x His (hexa histidine). However, the types of sequences necessary for purifying the target protein are not limited by the above examples. In the case of a fusion protein expressed by a vector containing the fusion sequence, it can be purified by affinity chromatography. For example, when glutathione-S-transferase is fused, glutathione, which is a substrate of the enzyme, can be used. When 6x His is used, Ni-NTA His-conjugated resin column (Novagen, USA) Can be easily recovered.
The stab nucleic acid molecule of the present invention can be used to increase the expression of a specific promoter in a variety of host cells, thereby helping to mass-express useful enzymes or proteins, and thus can be effectively used for the production of industrially useful proteins.
Figure 1 is a schematic representation of the plasmids pD3S0 and pD3CaS. Ap R, Sp R and Cm R represents an ampicillin, respectively, spectinomycin and chloramphenicol resistance genes. P cry3Aa means a cry3Aa promoter that does not contain a stab nucleic acid molecule.
FIG. 2 is a graph comparing the amount of beta-galactosidase expressed in the plasmid-containing strain shown in FIG. no stab means pD3S0.
Fig. 3 is a diagram showing the plasmids pDG-PSA3 and pDG-P4CaS. Ap R, Sp R and Cm R represents an ampicillin, respectively, and the spectinomycin resistance gene article L'cancer penny call. P phoB refers to the phoB promoter derived from Bacillus subtilis.
Fig. 4 is a graph comparing the amount of beta-galactosidase expressed in a plasmid-containing strain shown in Fig. 3; Fig.
FIG. 5 is a diagram illustrating the plasmids pAD-PSA3, pAP16 and pAP17. Ap R and Cm R represent the ampicillin and chloramphenicol resistance genes, respectively, and Rep1060 represents the origin of replication for Bacillus. P phoB is derived from Bacillus subtilis phoB Quot; promoter ".
FIG. 6 is a graph showing the expression amount of GFP in a strain containing the plasmid shown in FIG. 5 by a flow cytometer. FIG. The arrow indicates the expressed GFP peak.
Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are provided to illustrate the present invention more easily, and the scope of the present invention is not limited to these examples.
Example One: stab Securing nucleic acid molecules and using them cry3Aa Promoter Expression level increase
<1-1> Securing stab nucleic acid molecule
stab nucleic acid molecule (3CaS1) derived from cry3Ca was obtained by PCR using primers 3CaS-F (SEQ ID NO: 3) and 3CaS-R (SEQ ID NO: 4). The obtained stab nucleic acid molecule was digested with SpeI and BamHI and inserted into the SpeI and BamHI sites of the plasmid pD32 (Korean Patent Application No. 10-2008-0122155) to complete the plasmid pD3CaS (Fig. 1). Plasmid pD3S0 (FIG. 1) containing no stab nucleic acid molecule used as a control was prepared by digesting plasmid pD32 with SpeI and BamHI, making both ends blunt-ended with Klenow enzyme, and ligation. The prepared plasmids were introduced into Bacillus subtilis 168 (Kunst F. et al. 1997. Nature 390: 249-256) by natural selection method.
≪ 1-2 > Increased amount of cry3Aa promoter expression by stab nucleic acid molecule
Bacillus subtilis strains containing the plasmids pD3S0 and pD3CaS were inoculated into DSM medium (Harwood CR and Cutting SM, 1990. p549, Molecular Biological Methods for Bacillus, John Wiley & Sons Ltd.) And the activity of β-galactosidase was measured by a method using toluene (Harwood CR and Cutting SM, 1990, p443, Molecular Biological Methods for Bacillus, John Wiley & Sons Ltd.). As a result, 3CaS1 increased the expression amount of the cry3Aa promoter by about 10 times (FIG. 2).
Example 2: stab By nucleic acid molecule phoB Promoter Expression level increase
The stab nucleic acid molecule was applied to other promoters to see if the increase in the expression level by the stab nucleic acid molecule was confined to the cry3Aa promoter. To this end, the stab nucleic acid molecule was digested with SpeI and BamHI and inserted into the SpeI and BamHI sites of the vector pDG-PSA4 (Korean Patent Application No. 10-2008-0074253) with the Bacillus subtilis phoB promoter to obtain the plasmid pDG-P4CaS (Fig. 3). As a control, a plasmid pDG-PSA3 having a phoB promoter not containing a stab nucleic acid molecule was used (FIG. 3). Bacillus subtilis strains containing the plasmids pDG-PSA3 and pDG-P4CaS were inoculated into CLPYG medium (Choi SK and Saier MH, 2005. J. Mol. Microbiol. Biotechnol. 10: 40-50) The activity of beta-galactosidase was measured at 1 hour intervals using toluene (Harwood CR and Cutting SM, 1990, p443, Molecular biological methods for Bacillus, John Wiley & Sons Ltd.). As a result, 3CaS1 increased the expression amount of the phoB promoter by about 1.7 times (FIG. 4).
Example 3: The 5 'end of the structural gene Combined stab Securing nucleic acid molecules and using them phoB Promoter Expression level increase
<3-1> Securing the stab nucleic acid molecule bound to the 5 'end of the structural gene
Meanwhile, in the stab nucleic acid molecule 3CaS1 prepared in Example 1-1, the stab nucleic acid molecule 3CaS2 including the 5'-terminal part of the cry3Ca structural gene was amplified with the primer 3CaS-F3 (SEQ ID NO: 5) and the primer 3CaS- 6) was obtained from plasmid pAP16 by PCR. The plasmid pAP16 was completed by digesting the stab nucleic acid molecule 3CaS1 with SpeI and BamHI and then inserting it into the SpeI and BamHI sites of the plasmid pAD-PSA4 (Korean Patent Application No. 10-2008-0074253) (FIG. 5). The obtained stab nucleic acid molecule 3CaS2 was digested with SpeI and BamHI and inserted into the SpeI and BamHI sites of the plasmid pAD-PSA4 to complete the plasmid pAP17 (FIG. 5). Plasmid pAD-PSA3 containing no stab nucleic acid molecule used as a control was prepared by digesting plasmid pAD-PSA4 with SpeI and BamHI, blunt-ending both ends with Klenow enzyme, and ligation 5). The prepared plasmids were introduced into Bacillus subtilis 168 (Kunst F. et al. 1997. Nature 390: 249-256) by natural selection method.
≪ 3-2 > Increase in expression of phoB promoter by stab nucleic acid molecule
Bacillus subtilis strains including the plasmids pAD-PSA3, pAP16 and pAP17 were inoculated into CLPYG medium (Choi SK and Saier MH, 2005. J. Mol. Microbiol. Biotechnol. 10: 40-50) The expression level of GFP was analyzed by flow cytometry. As a result, 3CaS1 did not increase the expression amount of the phoB promoter, but 3CaS2 increased the expression amount of the phoB promoter by about 13 times (FIG. 5).
As described above, the stab nucleic acid molecules of the present invention can increase the expression amount of other promoters. In particular, it has been confirmed that the stab nucleic acid molecule containing the 5 'end of the structural gene can significantly increase the expression amount of another promoter, Of the present invention.
<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> NOVEL NUCLEIC ACIDS FOR ENHANCING GENE EXPRESSIONS AND METHOD FOR PROTEIN PRODUCTION USING THE SAME <130> PA100009 / KR <150> KR10-2009-0007587 <151> 2009-01-30 <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 129 <212> DNA <213> Bacillus thuringiensis <220> <221> 5'UTR <222> (1). (129) <223> 3CaS1 stab <400> 1 gtttgaaagg ggggatgtgt taaaagaaag aatattaaaa tcttgtgttt gtaccgtcta 60 atggatttat gggaaattat tttatcagat tgaaagttat gtattatgac aagaaaggga 120 ggaagaaaa 129 <210> 2 <211> 178 <212> DNA <213> Bacillus thuringiensis <220> <221> 5'UTR ≪ 222 > (1) .. (178) <223> 3CaS2 stab <400> 2 gtttgaaagg ggggatgtgt taaaagaaag aatattaaaa tcttgtgttt gtaccgtcta 60 atggatttat gggaaattat tttatcagat tgaaagttat gtattatgac aagaaaggga 120 ggaagaaaaa tgaatccgaa caatcgaagt gaacatgata caataaaagc tactgaaa 178 <210> 3 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Primer for 3CaS-F <400> 3 tatactagtg tttgaaaggg gggatgtgtt aaaagaaaga atattaaaat cttgtgtttg 60 taccgtctaa tggatttatg gga 83 <210> 4 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Primer for 3CaS-R <400> 4 tatggatcct tttcttcctc cctttcttgt cataatacat aactttcaat ctgataaaat 60 aatttcccat aaatccatta gac 83 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer for 3CaS-F3 <400> 5 tatactagtg tttgaaaggg gggatgtg 28 <210> 6 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> Primer for 3CaS-R3 <400> 6 tatggatcct ttcagtagct tttattgtat catgttcact tcgattgttc ggattcattt 60 ttcttcctcc ctttcttgtc 80
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