KR20130055481A - A method for tunable control of protein expression - Google Patents

Abstract

PURPOSE: A method for minutely regulating protein expression is provided to regulate expression pattern of the target protein using a vector comprising a promoter, a target protein-coding nucleotide sequence, and a random sequence, and to establish expression pattern of the target protein and relation of an introduced sequence. CONSTITUTION: A method for providing information of a triple random sequence for inserting a first sub-nucleotide and a second sub-nucleotide, which is able to regulate expression pattern of a target protein comprises: a step of preparing a vector containing a promoter, a target protein-coding nucleotide sequence, and the triple random sequence; a step of introducing the vector into a host cell; and a step of collecting 1:1 relation of expression pattern of the target protein produced from the host cell and the triple random sequence.

Description

A method for tunable control of protein expression

The present invention provides a vector comprising a promoter, a nucleotide sequence introduced in multiples of 3, and a protein-encoding nucleotide sequence for promoters to finely control the expression pattern of the protein of interest; A method of providing information of an introduced random nucleotide sequence capable of controlling the expression of a target protein by collecting a relationship with an expression amount of a target protein produced using the vector; And it relates to a method for producing a protein of interest using the vector.

Microorganisms are mainly used in the mass production of useful substances such as chemicals and enzymes or in the food fermentation industry. Bacillus, along with E. coli and yeast, has been used as a major host to produce industrially useful proteins. The benefits of Bacillus as a protein-producing host are harmless to the human body, can secrete large amounts of proteins extracellularly, do not produce endotoxins, and are regulated by the FDA as GRAS (generally regarded as safe) and codon biased. (codon bias), genetic engineering, transformation and mass cultivation are easy, and genomic information is known (Shumann, W., 2007, Adv . Appl. Microbiol ., 62: 137-189). Currently, about 60% of the world's industrial enzymes are produced from Bacillus as host bacteria (Westers, L., 2004, Biochimica). et Biophysica Acta , 1694: 299-310) It is known to be suitable for the production of food or pharmaceutical proteins due to its nature as a harmless GRAS microorganism (Schallmey, M., 2004, Can . J. Microbiol ., 50: 1217). . In order to produce a large amount of classically useful enzymes or proteins, a method of mutating a host bacterium through mutation inducer treatment, ultraviolet light, or irradiation has been used. It was reduced or wasted a lot of time and effort because it was necessary to develop a host bacteria for the production of each desired protein. Recently, however, a method of making recombinant microorganisms using genetic engineering techniques and using them to increase the productivity of a desired protein or substance has been attempted. Here, a method of inducing overexpression of a target protein using a strong promoter is the basis (Jana). , S. and Deb, JK, 2005 , Appl Microbiol Biotechnol, 67:...... 289-298; Shumann, W., 2007, Adv Appl Microbiol, 62: 137-189). However, overexpression of the protein of interest often results in the formation of an inclusion body to produce inactivated proteins, which requires an optimal control of the expression level of the protein of interest (Sorensen, HP and Mortensen). , KK, 2007, J. Biotechnol ., 115: 113-128). On the other hand, in the mass production of cell metabolites, a method of overexpressing a major enzyme during metabolism and inactivating an enzyme involved in unnecessary metabolic flow is mainly used. However, this "all or nothing" extreme approach often results in unstable cell viability, resulting in lower production of the target material (Snoep, JL et. al . , 1995, Microbiology , 141: 2329-2337. Therefore, in order to mass-produce a target substance while maintaining an optimal metabolic flow, a method capable of finely controlling the expression of a related enzyme is required.

The expression of genes is done by a promoter, which consists of -35 sites, -10 sites, and the spaces between them. The nucleotide sequence of the -35 site and the -10 site varies depending on the sigma factor of the RNA polymerase binding thereto. For example, the -35 region and -10 region of the promoter recognized by Sigma A have a consensus sequence of TTGACA and TATAAT, respectively. The space has no common sequence but has a constant length, and the space recognized by sigma A consists of 17 bases. Recently, the space region has been reported to influence the strength of the promoter (Jensen, PR and Hammer, K., 1998, Appl . Environ. Microbiol . , 64: 82-87), using this to finely control the strength of the promoter Have been developed (Solem, C. and Jensen, PR, 2002, Appl . Environ . Microbiol. , 68: 2397-2403; Hammer, K. et. al ., 2006, Trends in Biotechnol ., 24: 53-55). These techniques use a method of screening promoters of various intensities after constructing a promoter having a random nucleotide sequence in the space having a common -35 and -10 sites of a promoter recognized by sigma A. In addition, a technique has been developed to diversify promoters using molecular evolution techniques and then finely regulate protein expression (Alper, H. et. al ., 2005, PNAS , 102: 12678-12683). Most promoters are regulated by a variety of factors associated with the metabolic flow in the cell, thus expressing the protein in the right environment and time. The regulation associated with this metabolic flow helps the cell to maintain its optimal state. However, all of the protein expression microregulation techniques developed so far are altered upstream of the target protein gene, including the promoter, and thus may affect the expression of genes related to metabolic flow.

Therefore, the present inventors have made intensive studies to develop a technique capable of finely controlling protein expression without changing the upstream region of the target protein gene.As a result, the random sequence of the short length at the N terminus after the start codon of the target protein is multiplied by three. When the target protein was expressed in a form in which the peptide of the short length was bound to the N terminus, it was confirmed that the target protein was produced in various expression amounts. Accordingly, the present invention was completed by confirming that the expression level of the protein of interest can be finely controlled by inserting a random sequence into the frame after the start codon of the protein of interest.

One object of the present invention is to generate and generate a random multiple of 3 additionally introduced between a first subnucleotide sequence that is transcribed into an initiation codon of a protein-encoding nucleotide sequence and a second subnucleotide sequence that is transcribed into a second codon. Collecting a 1: 1 relationship of the expression pattern of the protein of interest to provide a method of providing the three-fold random sequence information of the three that can adjust the protein expression pattern (protein expression pattern of interest).

Another object of the invention is to use a vector comprising a multiple of three base sequences between a first subnucleotide sequence that is transcribed into an initiation codon of a protein-encoding nucleotide sequence and a second subnucleotide sequence that is transcribed into a second codon It is to provide a method for producing a protein.

Another object of the present invention is to express the target protein by adding a multiple of 3 base sequences between the first subnucleotide sequence to be transcribed into the start codon of the protein-encoding nucleotide sequence and the second subnucleotide sequence to be transcribed to the second codon. It is to provide a vector for controlling the aspect.

In one aspect to achieve the above object, the present invention provides a method for the production of (i) a promoter, (ii) a protein-coding nucleotide sequence, and (iii) a first subnucleotide transcribed into an initiation codon of the protein-coding nucleotide sequence. Preparing a vector comprising a random nucleotide sequence of multiples of 3 additionally introduced between the sequence and a second subnucleotide sequence that is transcribed into a second codon; A second step of introducing the prepared vector into a host cell; And a third step of collecting a 1: 1 relationship between the expression pattern of the protein of interest produced from the host cell and the random nucleotide sequence of three folds introduced in the first step, the protein expression pattern of protein Provided is a method of providing three-fold random sequencing information for insertion between a first subnucleotide and a second subnucleotide capable of controlling interest.

In another embodiment, the present invention provides a second subdon and a first subnucleotide sequence transcribed into (i) a promoter, (ii) a protein-coding nucleotide sequence of interest, and (iii) an initiation codon of the protein-coding nucleotide sequence of interest. A first step of preparing a vector comprising a nucleotide sequence of multiples of 3 additionally introduced between a second subnucleotide sequence to be transcribed into; It provides a method for producing a protein of interest comprising the second step of producing the protein of interest by introducing the prepared vector into the host cell.

In another embodiment, the present invention provides a second subdon and a first subnucleotide sequence transcribed into (i) a promoter, (ii) a protein-coding nucleotide sequence of interest, and (iii) an initiation codon of the protein-coding nucleotide sequence of interest. It provides a target protein expression modulating vector comprising a nucleotide sequence of multiples of 3 additionally introduced between the second subnucleotide sequence to be transcribed into.

The random nucleotide sequence additionally introduced into the protein-coding nucleotide sequence is (i) a DNA sequence ATG transcribed between the first subnucleotide sequence that is transcribed into the start codon and the second subnucleotide sequence that is transcribed into the second codon, i.e. It is inserted immediately after, and (ii) is inserted in multiples of three. Thus, the cell is maintained in an optimal state because it does not modify the promoter associated with metabolic flow. In addition, since the gene to be transcribed into the start codon of the target protein is inserted in the same frame as the gene of the target protein in multiples of 3, the target protein is expressed in a form in which a short-length peptide is bound to the N-terminus. The results were shown to vary the expression pattern.

The third step is characterized by identifying a random nucleotide sequence of multiples of 3 inserted by sequencing. Sequencing may be performed using Sanger chain termination sequencing, sequencing, automatic sequencing using PCR, pyrosequencing, analysis using DNA chips, and the like. The method can be used without limitation.

By expressing the protein of interest using a vector in which a random nucleotide sequence of three folds was introduced immediately after the DNA sequence ATG transcribed into the start codon by the method of the present invention, cells having a desired expression pattern were selected by searching for the expression pattern thereof. By doing so, it is possible to produce a protein whose expression level is adjusted to the purpose.

The term "promoter" of the present invention is a region on DNA that promotes transcription of a particular gene. That is, as a non-translated DNA sequence having a binding site for an RNA polymerase or transcription factor, the promoter is located close to the gene to be controlled and is on the same strand and is generally upstream, that is, sense It is in the 5 'region of the sense strand. In eukaryotes, proteins called transcriptional regulators are involved in the induction of RNA polymerase by binding to the promoter moiety. Promoters can be said to refer to all the DNA sequence to which these transcription regulators bind. By contrast, in prokaryotes, it is usually defined as the binding site near the transcription start site to which the RNA polymerase binds. In prokaryotes, the promoter consists of two short bases, 10 base pairs and 35 base pairs, each forward from the start of transcription. The base sequence in front of the 10 base pair is called a pribnow box or -10 element (-10 element, -10 site) and is generally composed of six base sequences of TATAAT. Privno boxes are essential for initiating transcription in prokaryotes. The base sequence in front of the 35 base pair (-35 site) usually consists of six base sequences of TTGACA and helps to facilitate transcription. In the embodiment of the present invention, since Bacillus bacteria are used as host cells, any promoter capable of expressing Bacillus bacteria may be used without limitation. Preferably, it is possible to synthesize and use a promoter P5D having an improved function compared to a natural promoter. The synthesis can be carried out by a variety of methods known in the art, preferably by performing PCR using a variety of plasmids and primers to obtain a PCR product, and then mixed again to synthesize by PCR to obtain the final target promoter have.

The term "vector" of the present invention is an expression vector capable of expressing a protein of interest in a suitable host cell, and refers to a gene construct comprising essential regulatory elements operably linked to express the gene insert.

In general, plasmid vectors are cyclic double-stranded DNA extracellularly and present in cells to serve a variety of functions. It produces antibiotics and bacteriocins, and acts as an inhibitor to kill similar strains or species, and performs physiological functions such as pigment formation, compound degradation, and nitrogen fixation. It has a restriction site to insert foreign DNA fragments up to about 10 kb in length. The ability to insert only relatively small DNA fragments, a major drawback of plasmids and bacteriophages used as vectors, is to clone larger DNA fragments using cosmids, engineered hybrids of plasmid and phage DNA. There is a cos site that is packaged into phage particles and has a starting point for replication of the plasmid that replicates in the bacterial host and a gene that can select the plasmid. Although packaged in vitro as a protein envelope, such as bacteriophage phage vectors, after the packaged DNA infects E. coli host cells, the DNA replicates in plasmid form rather than bacteriophage DNA and does not lyse. When the cos site is separated from 37 to 52 kb in size after 2.5 kb of infected cells, the foreign DNA is inserted into the insert. Generally 35 to 45 kb can be cloned into cosmid vectors. Λ phage, a common form of vector of bacteriophage, is also derived from a 50 kb double-stranded wild genome with cohesive termini or cos with complementary ends of a single strand of 12 nucleotides capable of base pairing. In the lytic pathway, host cells are fused after the new virus has replicated and the progeny virus has been released. This type of DNA can add 3 kb to its total 52 kb, or accommodate only 5% of its genome, and a vector that makes room for foreign DNA is free of non-essential fragments of DNA. Viral vectors are retroviruses such as Human immunodeficiency virus HIV (Murine leukemia virus) Avian sarcoma / leukosis (ASLV), Spleen necrosis virus (SNV), Rus sarcoma virus (RSV) and Mouse mammary (MMTV). tumor viruses), including but not limited to vectors derived from Adenovirus, Adenoassociated virus, Herpes simplex virus, and the like. In a preferred embodiment of the present invention, the vector was prepared by cutting the plasmid pDG1661 using EcoRI and BsiWI and inserting the P5D-Lgfp variant sequence.

In the present invention, "operably linked" means that the promoter variant nucleic acid sequence comprising differently combined portions of the N-terminal sequence of the structural gene and the nucleotide sequence encoding the desired protein are functional to perform a general function. It is connected to. 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 non-translated nucleic acid sequence that aids 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 the promoter variants of the present invention as regulatory elements, expression control sequences that may affect protein expression, such as initiation codons, termination codons, polyadenylation signals, enhancers, membranes Signal sequences for targeting or secretion, and the like. Polyadenylation signals increase the stability of transcripts or facilitate cellular transport. 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 α-amylase signal sequence and subtilisin signal sequence when the host is Bacillus. In this case, an MFα signal sequence, a SUC2 signal sequence, or the like may be used, but is not limited thereto. In addition, 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 nucleic acid sequence encoding the protein of interest with the vector is inserted and expressed downstream of the promoter. The target protein insertable into the vector is not particularly limited and may insert various proteins for medical or industrial purposes. Medically and industrially useful proteins include hormones, hormone analogs, cytokines, enzymes, inhibitors, transport proteins, receptors and fragments of receptors, antigens, antibodies and antibody fragments, structural proteins, regulatory proteins, transcription factors, toxin proteins, biological Defensive inducers; In a preferred embodiment of the present invention, by operably linking GFP (green fluorescence protein) downstream of the variant promoter sequence to prepare a vector, the protein expression activity of the Bacillus strain including the vector was measured (Fig. 2).

As used herein, 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. In a host cell that can accept foreign DNA, foreign DNA such as a vector can be inserted into the host cell. When the vector is successfully introduced into the host cell, the host cell is provided with the genotype of the vector. . Preferably, the host cell of the present invention may be a Gram-positive bacterium, and the Gram-positive bacterium is a genus Bacillus, Lactobacillus genus, Brevibacillus genus, Penivacillus genus, Clostridium genus, Corynebacterium genus or Streptomyces It may be a genus bacterium. In this case, the bacterium of the genus Bacillus may be one selected from the group consisting of Bacillus cactilli, Bacillus rikenimomis, Bacillus megaterium, Bacillus thuringiensis or Bacillus sirius, but the vector of the present invention is transformed Possible host cells are not limited thereto. Preferably, in the present invention, Bacillus certilis was used as the host cell, and more preferably, Bacillus certilis 168 was used to confirm the expression of the target protein.

As a method of introducing a vector into a host cell, a transformation method may be used. "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 can be used as the transformation method and can be easily carried out according to a conventional method in the art. In general, the transformation method uses the CaCl ₂ precipitation method, the CaCl ₂ method, a Hanahan method that increases efficiency by using a reducing material called DMSO (dimethyl sulfoxide), electroporation, calcium phosphate precipitation, plasma fusion method, silicon carbide Agitation with fibers, agrobacterial mediated transformation, transformation with PEG, dextran sulfate, lipofectamine and dry / inhibition mediated transformation, and the like, but are not limited thereto. The transformation method used can be used without limitation.

In addition, the host cell transformed by the above method may be cultured through a culture method commonly used in the art as needed, and the medium and the culture period that can be used may be arbitrarily selected by those skilled in the art as needed. Preferably, the present invention is transformed Bacillus strain in TSB medium and incubated for 24 hours at 37 ℃ to induce the production of the protein of interest. The present invention may further comprise the step of recovering the protein of interest in order to measure the amount of the protein of interest produced through the culture.

In order to facilitate the purification of the protein of interest to be recovered in the present invention, other 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 (hexahistidine), but the examples do not limit the type of sequence required for purification of the protein of interest. In the case of the fusion protein expressed by the 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 this enzyme, can be used, and in the case of 6x His, a desired target protein can be obtained by using a Ni-NTA His-binding resin column (Novagen, USA). It can be easily recovered.

A multiple of 3 additionally introduced between the promoter of the invention, the target protein-coding nucleotide sequence and the first subnucleotide sequence to be transcribed into the start codon of the target protein-coding nucleotide sequence and the second subnucleotide to be transcribed to the second codon The vector comprising a random nucleotide sequence of can be used to control the expression of the target protein, and sequencing can be used to establish the relationship between the expression of the target protein and the introduced nucleotide sequence. At this time, the base sequence to be introduced is introduced in the multiplex of 3 after the start codon, thereby controlling the expression level of the target protein without changing the target protein gene upstream.

1 is a schematic diagram showing 30 nucleotide sequences inserted between the P5D promoter and the N terminus of the protein of interest.
FIG. 2 is a diagram analyzing the GFP expression level of Bacillus sertilis 168 strain in which a plasmid library having a combination of a P5D promoter and a gfp gene having 30 nucleotide sequences inserted into the N-terminal is introduced.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention more specifically and that the scope of the present invention is not limited by these embodiments.

Example  One: GFP  Fine regulation of expression

In order to finely regulate the expression of GFP, a variant (Lgfp) was prepared in which 30 random sequences were fused to the N-terminus of the gfp gene in the same frame as the gfp gene (FIG. 1). To this end, gfp was PCR from plasmid pAD123 (Dunn, AK and Handelsman, J., 1999, Gene , 226: 297-305) using primers Gfp-Lf (SEQ ID NO: 1) and gfp-r1 (SEQ ID NO: 2). Secured. Promoter P5D is a PCR product and plasmid pD52 (Lee, S.-J) obtained from plasmid pA84 (Korean Patent 10-2010-0010044) using primers 3Aag-f2 (SEQ ID NO: 3) and P83-R1 (SEQ ID NO: 4). et al ., 2010, J. Biotechnol. , 149: 16-20), PCR products obtained by using primers P52-F1 (SEQ ID NO: 5) and stabr2 (SEQ ID NO: 6) were mixed with each other to perform a PCR reaction to obtain a promoter P5D, which is the final PCR product. . Secured Lgfp was again PCR with promoter P5D to secure P5D-Lgfp. P5D-Lgfp was digested with EcoRI / BsiWI and then plasmid pDG1661 (Guerout-Fleury, AM et al, 1996, Gene, 180: . and cloned into the same sites of 57-61), thereby completing the pD5D-Lgfp library.

The prepared plasmid library was introduced into Escherichia coli and plated in LBagar (Luria-Bertani Agar) medium containing 100 μg / ml of ampicillin. The plasmids were collected by collecting all the E. coli transformants formed on the agar medium, and the isolated plasmid library was Bacillus sertilis. subtilis ) 168 strain (Kunst, F. et al ., 1997, Nature , 390: 249-256). The produced Bacillus transformants were randomly selected 96 fluorescence strains, and then inoculated in TSB medium (Difco) and incubated at 37 ° C for 24 hours. Cultured strains were measured for expression of GFP using EnVision Multilabel Plate Readers (Perkin Elmer). As a result, it was confirmed that the expression level of GFP showed various distributions (FIG. 2).

Therefore, inserting a nucleotide sequence of a certain length into the N-terminus of the target protein gene in the same frame as the target protein gene so that the random peptide fragment is coupled to the N-terminus of the target protein, the expression of the target protein is varied and accordingly, It can be used for metabolic engineering or protein production by selecting the vector with excitation.

<110> Korea Research Institute of Biocience and Biotechnology <120> A method for tunable control of protein expression <130> PA110884 / KR <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Gfp-Lf <400> 1 gataagaaag ggaggaagaa aaatgnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnagtaa 60 aggagaagaa c 71 <210> 2 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> gfp-r1 <400> 2 tttcgctcgg gaagacgtac gttatttgta tagttcatcc a 41 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> 3Aag-f2 <400> 3 agctgtcaaa catgagaatt cgagctctca gcagtagaag 40 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> P83-R1 <400> 4 cacttttatc taaggtttc 19 <210> 5 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> P52-F1 <400> 5 gaaaccttag ataaaagtgc tttttttgtt gacattgaag aattattaat gttaagctta 60 attaaagata atatctttga attg 84 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> stabr2 <400> 6 ttttcttcct ccctttctta tcataata 28

Claims (9)

between (i) the promoter, (ii) the target protein-coding nucleotide sequence, and (iii) the first subnucleotide sequence that is transcribed into the initiation codon of the target protein-coding nucleotide sequence and the second subnucleotide sequence that is transcribed into the second codon. A first step of preparing a vector including a random nucleotide sequence of multiples of 3 additionally introduced into the;
A second step of introducing the prepared vector into a host cell; And
Including a third step of collecting a 1: 1 relationship between the expression pattern of the target protein produced from the host cell and the random nucleotide sequence of a multiple of 3 introduced in the first step,
A method of providing three fold random sequence information for insertion between a first subnucleotide and a second subnucleotide capable of controlling a protein expression pattern of interest.
The method of claim 1,
The third step is to provide a random sequence of multiples of 3 inserted by the sequencing method providing.
between (i) the promoter, (ii) the target protein-coding nucleotide sequence, and (iii) the first subnucleotide sequence that is transcribed into the initiation codon of the target protein-coding nucleotide sequence and the second subnucleotide sequence that is transcribed into the second codon. A first step of preparing a vector comprising a nucleotide sequence of multiples of 3 introduced additionally; And
Including the second step of producing the protein of interest by introducing the prepared vector into the host cell,
Method of production of protein.
The method of claim 3,
between (i) the promoter, (ii) the target protein-coding nucleotide sequence, and (iii) the first subnucleotide sequence that is transcribed into the initiation codon of the target protein-coding nucleotide sequence and the second subnucleotide sequence that is transcribed into the second codon. Preparing a vector comprising a random nucleotide sequence of multiples of 3 introduced further into;
Introducing the prepared vector into a host cell;
Selecting a cell having a desired expression pattern by searching for expression aspects of the protein of interest produced from the host cell; And
Culturing the selected cells to produce the protein of interest,
Method of production of protein.
5. The method according to any one of claims 1 to 4,
The host cell is a gram positive bacterium.
5. The method according to any one of claims 1 to 4,
The Gram-positive bacterium is a bacterium of the genus Bacillus, Lactobacillus, Brevibacillus, Penibacilli, Clostridium, Corynebacterium or Streptomyces.
5. The method according to any one of claims 1 to 4,
The bacterium of the genus Bacillus is one selected from the group consisting of Bacillus Cactilli, Bacillus rickeniformis, Bacillus megaterium, Bacillus thuringiensis or Bacillus sirius.
5. The method according to any one of claims 1 to 4,
The target proteins include hormones, hormone analogs, cytokines, enzymes, inhibitors, transport proteins, receptors and fragments of receptors, antigens, antibodies and antibody fragments, structural proteins, regulatory proteins, transcription factors, toxin proteins and biodefense inducers. At least one target protein selected from the group consisting of:
between (i) the promoter, (ii) the target protein-coding nucleotide sequence, and (iii) the first subnucleotide sequence that is transcribed into the initiation codon of the target protein-coding nucleotide sequence and the second subnucleotide sequence that is transcribed into the second codon. A vector for controlling the expression pattern of the protein comprising a base sequence of multiples of 3 introduced additionally.

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