KR101671626B1 - A novel expression cassette for secretion of protein - Google Patents

Abstract

More particularly, the present invention relates to a novel expression cassette for a target protein fraction based on microorganisms of the genus Corynebacterium, and a method for producing a target protein for use therefor.

Description

A novel expression cassette for secretion of protein < RTI ID = 0.0 >

More particularly, the present invention relates to a novel expression cassette for a target protein fraction based on a microorganism of the genus Corynebacterium, and a method for producing a target protein.

Corynebacterium sp. Microorganisms are widely used in industrial production of amino acids, as well as in production of major recombinant proteins in the pharmaceutical, food and chemical industries. This is because microorganisms of the genus Corynebacterium have ideal characteristics as recombinant protein production platforms. For example, i) there is very little protein secreted out of the cell, ii) there is little proteolytic activity outside the cell, and iii) there is only one cell membrane because of Gram-positive bacterium, (Alain Vertes; Springer; Protein secretion systems of Corynebacterium glutamicum). On the other hand, industrial production rate, production yield, quality, and functionality of recombinant protein are important factors in the production of recombinant proteins. Recently, studies on the production of recombinant proteins have been carried out in order to improve the promoter of target proteins, codon optimization, translational signals engineering and production of byproducts, or to improve host strains so as to use cheap raw materials. In particular, Gram-positive bacterium has various sugar availability such as glucose, raw sugar, and fructose. It can cultivate high concentration cells and selects high-efficiency secretion pathway through transcription and translation control. (Long Liu et al., Appl. Microbiol Biotechnol (2013) 97: 9597-9608), which secrete the target protein outside the cell to produce an industrially recombinant protein. However, there is still a need for the development of new, fractionally expressible cassettes that can be used in microorganisms of the genus Corynebacterium.

Under these circumstances, the present inventors have made intensive efforts to develop a cassette for expression of a novel protein capable of over-secretion of a target protein in a microorganism belonging to the genus Corynebacterium. As a result, they have developed a novel secreted peptide, a signal peptide and its modified form, The present invention has been completed by constructing a cassette expressing the cost of a recombinant protein.

One object of the present invention is to provide a novel target protein cost expression cassette which comprises (i) a promoter; (ii) a base sequence encoding a signal peptide having an amino acid sequence represented by SEQ ID NO: 1 or 2; And (iii) a gene encoding a target protein.

Another object of the present invention is to provide a separated signal peptide having the amino acid sequence of SEQ ID NO: 1 or 2 which is a novel signal peptide and a nucleic acid molecule encoding the same.

It is another object of the present invention to provide a vector comprising the nucleic acid molecule encoding the expression cassette or the signal peptide.

Another object of the present invention is to provide a microorganism belonging to the genus Corynebacterium, into which the expression cassette or vector expressing the desired protein fraction is introduced.

It is another object of the present invention to provide a method for producing a target protein using the microorganism of the genus Corynebacterium.

In order to achieve the above object, the present invention provides, in one aspect, a cassette for expression of a target protein, which is a novel secretion system. The expression cassette for cost-of-interest of interest comprises (i) a promoter; (ii) a base sequence encoding a signal peptide having an amino acid sequence represented by SEQ ID NO: 1 or 2; And (iii) a gene encoding a target protein.

Specifically, the cassette for expressing a target protein is specifically a plasmid in which a base sequence encoding a signal peptide having an amino acid sequence represented by SEQ ID NO: 1 or 2 is operably linked downstream of the promoter sequence, Gene. ≪ / RTI >

The expression cassette is characterized in that the secretion ability of the target protein is improved in the microorganism of the genus Corynebacterium as compared with the expression cassette which does not contain the nucleotide sequence encoding the signal peptide of SEQ ID NO: 1 or SEQ ID NO: 2. In the present invention, the term "promoter " refers to a nucleotide sequence that is upstream of the coding region and contains a binding site for a polymerase and has a transcription initiation activity to the mRNA of the promoter sub-gene. That is, it is a DNA region that allows the polymerase to bind and start transcription of the gene, and is located at the 5 'site of the mRNA transcription initiation site.

In the present invention, the term "signal peptide" can be used in combination with a "secretory peptide ", and a short peptide present at the N-terminus of the protein such that newly synthesized protein is secreted through a secretory pathway 5 to 30 amino acids). For the purpose of the present invention, the signal peptide may include any signal peptide as long as it is designed to secrete the desired protein outside the cell, but may be, for example, the amino acid sequence of SEQ ID NO: 1 or 2.

Specifically, the signal peptide has not only the amino acid sequence of SEQ ID NO: 1 or 2 but also a signal peptide having 80%, 90%, 95%, 96%, 97%, 98% or 99% . ≪ / RTI > In addition, as long as it is an amino acid sequence which exhibits the same or corresponding secretory activity as that of the above-mentioned signal peptide, as well as the signal peptide having the above homology, it includes without limitation. It is also apparent that amino acid sequences in which some sequences are deleted, modified, substituted or added are also included within the scope of the present invention, provided that they have such homology.

The nucleotide sequence coding for the signal peptide may be any nucleotide sequence which is substantially identical to the signal peptide or is encoded by a peptide exhibiting the corresponding activity at the time of coding. Specifically, it may be a nucleic acid molecule of SEQ ID NO: 3 or SEQ ID NO: 4, and it may be more than 80%, specifically more than 90%, more specifically more than 95%, more specifically more than 98% May be a base sequence having 99% or more homology. Also, it is obvious that base sequences having deletion, modification, substitution or addition of some sequences are also included within the scope of the present invention if they are base sequences having such homology. It may be termed a signal sequence and may include additional base sequences in addition to codon sequences that match 1: 1 with the signal peptide.

As used herein, the term "homology" refers to the percentage of identity between two polynucleotide or polypeptide mimetics. The homology between sequences from one moiety to another can be determined by known techniques. For example, homology can include sequence information between two polynucleotide molecules or two polypeptide molecules, such as score, identity, and similarity, using a computer program that aligns sequence information and is readily available. (E.g., BLAST 2.0). ≪ / RTI > In addition, homology between polynucleotides can be determined by hybridizing the polynucleotides under conditions that result in a stable double strand between homologous regions, then resolving the single-strand-specific nucleases to determine the size of the resolved fragment.

In the present invention, the term "target protein" or "target product" means a protein or product secreted from a microorganism or produced in large quantities using microorganisms. Specifically, it may include, without limitation, any protein or product secreted from, or produced by, a microorganism belonging to the genus Corynebacterium.

The term "expression cassette " in the present invention includes a base sequence encoding a promoter and a signal peptide, and a gene encoding a target protein, so that a target protein operably linked downstream of the signal peptide is expressed The unit cassette can be used. In the present invention, the expression cassette for division can be mixed with the secretion system. Inside or outside of such an expression cassette, various factors capable of helping efficient production of the target protein can be included.

The term "operably linked" in the present invention means that the nucleotide sequence having the promoter activity of the present invention and / or the nucleotide sequence coding for the signal peptide is expressed in the gene sequence and the promoter And / or a base sequence encoding a signal peptide is functionally linked. Operable linkages can be made using known recombinant techniques in the art, and site-specific DNA cleavage and linkage can be made using, but not limited to, cutting and linking enzymes in the art.

In another aspect, the present invention provides a novel signal peptide. Specifically a separate signal peptide having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

The signal peptide is as described above.

According to a specific embodiment of the present invention, within the N-terminus of the Cgl1342 protein identified to be a hypothetical protein of unknown function, the signal peptide of SEQ ID NO: 1 was isolated. Further, a signal peptide having the amino acid sequence of SEQ ID NO: 2 in which leucine (Leu, L), which is the first amino acid of the amino acid sequence of SEQ ID NO: 1, was substituted with methionine (Met, M) was isolated as an improved variant. It was confirmed that the above-mentioned signal peptide secreted the target protein, specifically xylanase, irrespective of the change of the promoter at the upper end (Fig. 5), and it was confirmed that the xylanase can be mass-secreted under the high concentration culture condition ).

In another aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned signal peptide.

The nucleic acid molecule encoding the signal peptide is as described above.

In another aspect, the present invention provides a vector comprising the expression cassette or a nucleic acid molecule encoding the signal peptide.

The vector comprising the nucleic acid molecule encoding the signal peptide may further comprise a gene encoding a target protein and the gene may be operably linked to a nucleic acid molecule encoding the signal peptide in the vector .

The term "vector" of the present invention refers to an artificial DNA molecule having a genetic material so that it can express a gene of interest in a suitable host strain. Specifically, a DNA preparation containing a base sequence of a gene operably linked to a suitable regulatory sequence it means. The regulatory sequence may include one or more of a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation But is not limited thereto.

The vector used in the present invention is not particularly limited as long as it is replicable in the host cell, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in their natural or recombinant state. For example, pWE15, M13, λMBL3, λMBL4, λXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as the phage vector or cosmid vector, and pBR, pUC, pBluescriptII , pGEM-based, pTZ-based, pCL-based, pET-based, and the like. The vector usable in the present invention is not particularly limited, and known expression vectors can be used. For example, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pCES208 vectors and the like can be used.

In addition, a sequence encoding an intrinsic signal peptide in a chromosome can be replaced with a nucleic acid molecule that is a sequence encoding a signal peptide of the present invention, or inserted into a sequence that does not contain an intrinsic signal peptide, through a vector for insertion of a chromosome in a host cell . The insertion of the nucleic acid molecule into the chromosome can be accomplished by any method known in the art, for example, homologous recombination. Since the vector of the present invention can be inserted into a chromosome by causing homologous recombination, it may further include a selection marker for confirming whether or not the chromosome is inserted. Selection markers are used to select cells that are transfected with a vector, that is, to confirm the insertion of a target nucleic acid molecule, and are provided with selectable phenotypes such as drug resistance, resistance to nutritional requirement, tolerance to cytotoxic agents, May be used. In the environment treated with the selective agent, only the cells expressing the selectable marker survive or express different phenotypes, so that the transformed cells can be selected.

Therefore, even in the case of a vector in which a desired gene is not operably linked to a nucleic acid molecule having a base sequence coding for a signal peptide having the amino acid sequence of SEQ ID NO: 1 or 2 of the present invention, the host cell may be a host cell such as Corynebacterium May be replaced by homologous recombination with an endogenous signal peptide in the genus Microorganism, or inserted by homologous recombination into a promoter and an endogenous protein. Whereby the endogenous gene of the microorganism of the genus Corynebacterium can be secreted and expressed.

In another aspect of the present invention, there is provided a microorganism belonging to the genus Corynebacterium into which the above-described vector is introduced, or a cassette expressing the desired protein fraction.

The expression cassette or vector expressing the target protein fraction can be introduced into the microorganism of the genus Corotte bacteria by transformation. For the purpose of the present invention, the term "transformed" in the present invention means that, for the purpose of the present invention, a vector comprising a cassette expressing the desired protein component or a nucleic acid molecule encoding a signal peptide or a cassette for expressing a protein of interest is introduced into the host cell, Quot; means that the nucleic acid molecule encoding the expression cassette or signal peptide can be expressed. The target protein can be expressed by integrating a portion of the cassette with the same sequence as a portion of the chromosome of the Corynebacterium microorganism on both sides of the cassette and integrating the entire expression cassette for expression of the desired protein at a specific position to achieve expression and secretion of the desired protein . A gene encoding a nucleic acid molecule encoding a signal peptide contained in the expression cassette or vector and / or a gene encoding a target protein operably linked thereto may be inserted into the chromosome of the host cell, Lt; RTI ID = 0.0 > chromosomal < / RTI > In addition, the nucleic acid molecule may include DNA and RNA in the form of a gene encoding a target protein. The expression cassette or vector may be introduced in any form as long as it can be introduced into a host cell and expressed. For example, an expression cassette, which is a gene construct containing all the elements required to be expressed by itself, may further include a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. The expression cassette may also be introduced into the host cell in its own form and operably linked to the sequence necessary for expression in the host cell.

The method of transforming the vector of the present invention includes any method of introducing a nucleic acid into a cell and may be carried out by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, A lithium acetate-DMSO method, and the like, but are not limited thereto.

The host cell may be any microorganism as long as the DNA introduction efficiency is high and the efficiency of the introduced DNA is high, but it may be a microorganism belonging to the genus Corynebacterium for the purpose of the present invention. Specifically, it may be, but is not limited to, Corynebacterium glutamicum.

In another embodiment of the present invention, there is provided a method for producing a target protein by secretory production of a target protein by culturing the microorganism of the genus Corynebacterium to which the vector containing the expression cassette of the present invention or the nucleic acid molecule encoding the signal peptide is introduced, And recovering the secreted protein from the microorganism or culture medium. The term "cultivation" in the present invention means cultivation of microorganisms under moderately artificially controlled environmental conditions. In the present invention, a method for producing a target product using a microorganism belonging to the genus Corynebacterium can be carried out by a method well known in the art. Specifically, the culturing can be carried out continuously in a batch process, an injection batch, or a fed batch or repeated fed batch process, but is not limited thereto. As a specific example, it may be a high-concentration cell culture through fed-batch culture.

The medium used for the culture should meet the requirements of the particular strain in an appropriate manner. Culture media for Corynebacterium sp. Strains are known (see, for example, Manual of Methods for General Bacteriology, American Society for Bacteriology, Washington D.C., USA, 1981). Sugar sources that may be used include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil and the like, palmitic acid, , Fatty acids such as linoleic acid, alcohols such as glycerol, ethanol, organic acids such as gluconic acid, acetic acid, and pyruvic acid, but are not limited thereto. These materials may be used individually or as a mixture. Examples of nitrogen sources that may be used include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, But is not limited thereto. The nitrogen source may also be used individually or as a mixture. The number of people that can be used includes, but is not limited to, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts. In addition, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth. Finally, in addition to these materials, essential growth materials such as amino acids and vitamins can be used. In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in a batch manner or in a continuous manner by an appropriate method. Such a variety of cultivation methods are disclosed, for example, in "Biochemical Engineering" by James M. Lee, Prentice-Hall International Editions, pp 138-176.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture. In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. An oxygen or oxygen-containing gas (e.g., air) can be injected into the culture to maintain aerobic conditions. The temperature of the culture may be usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C, but is not limited thereto.

A method for producing a target protein or a target product of the present invention may include recovering a target protein or a product from a microorganism or a culture and a method for recovering a target protein or product from the microorganism or culture is known in the art Methods such as centrifugation, filtration, anion exchange chromatography, crystallization and HPLC can be used, but the present invention is not limited to these examples.

The recovering step may include a purification step, and a person skilled in the art can select and utilize it according to the needs of various known purification processes.

Using the newly developed protein secretion cassette (i.e., a recombinant protein secretion system) of the present invention, a much stronger gene expression effect can be exhibited in a high concentration culture condition than in a gene expression system used in conventional Corynebacterium sp. Microorganisms have. Therefore, the target protein can be produced at a higher yield by using the Corynebacterium sp. Microorganism into which the recombinant protein secretion system of the present invention is introduced.

FIG. 1 is a schematic diagram of a protein secretion system in which a signal peptide is operably linked downstream of a promoter sequence and a gene encoding a target protein is downstream of the sequence.
FIG. 2 is a graph showing the conditions for culturing cells at a high concentration through fed-batch culture of Corynebacterium glutamicum for the present invention. Cell growth was expressed in black circles and glucose concentration in white circles.
FIG. 3 is a graph showing the secretory proteome (Secretome) of Corynebacterium glutamicum under high cell density culture conditions through 2DE (two-dimensional electrophoresis) analysis for the present invention. The circled portion is the protein spot identified as Cgl1342 protein through MALDI-TOF analysis.
FIG. 4 is an SDS PAGE analysis chart for confirming the secretion of Cgl1342 protein identified through proteome analysis of FIG. 3. FIG. The position of the marker portion of the triangle indicates the target protein containing the signal peptide, i.e., the signal peptide-Cgl1342.
FIG. 5 is a graph showing the secretion of a target protein, xylanase, using a secretion system comprising the cgl1342 gene promoter and the signal peptide of SEQ ID NO: 1 or SEQ ID NO: 2. (a) shows the amount of xylanase secreted by SDS PAGE analysis, and (b) shows the value obtained by measuring xylanase activity (Absorbance 550).
6 shows the results of production of xylalanase fermentation using a promoter of cgl1342 gene and a Corynebacterium glutamicum strain producing xylenase expressed by a protein secretion system using the signal peptide of SEQ ID NO: 2 . (a) shows the cell growth curve after fermentation and the glucose concentration in the medium (cell growth curve: (); glucose concentration: ()) and (b) shows the result of SDS- .
Figure 7 shows an example of a single chain antibody variable region gene (scFv) in a protein secretion system comprising various promoters and a signal peptide of SEQ ID NO: 1 or SEQ ID NO: 2 (denoted as "SS" and "SS1" (Absorbance 450) of the antibody fragments identified by ELISA analysis as the target protein.
8 is a comparative analysis of the protein secretion system disclosed in the literature and the protein secretion system discovered in the present invention. (a) is the analysis of the amount of the antibody fragment (scFv) secreted by SDS PAGE. (b) is a table showing the activity measurement value (Absorbance 450) of the antibody fragments identified through ELISA analysis.
9 is a diagram showing the results of fermentation of antibody fragments using Corynebacterium glutamicum strains producing antibody fragments by the secretion system using the cgl1342 promoter and the signal peptide of SEQ ID NO: 2. (a) shows the cell growth curve after fermentation and the glucose concentration in the medium (cell growth curve: (); glucose ()), and (b) shows the Western blot experiment result of the fermentation liquid obtained over time to be.

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

Example  One: Oil price formula  Through culture Corynebacterium Glutamicum  High cell culture

As a representative example of Corynebacterium sp. Microorganisms, a fed-batch culture is carried out under 5 L fermentation conditions to obtain a high cell density culture solution of Corynebacterium glutamicum wild type ATCC13032 strain Respectively. The ATCC13032 strain was streaked on a BHI (Brain Heart Infusion, Difco Laboratories) plate and incubated overnight in a 30 ° C incubator. One platinum was inoculated into a 500 ml baffle flask containing 50 ml of BHI liquid medium and incubated at 30 ° C. and 200 rpm for 24 hours Respectively. The cultured flask culture was inoculated with seed for 5L fermentation. 5L fermenter (BioCNS Co.) Operating conditions are shown in Table 1 below.

Medium composition _{40g glucose, 3 g K 2 HPO} 4, 1 g KH 2 PO 4, 2 g Urea, 10 g (NH4) 2 SO 4, 2 g MgSO 4, 200 ㎍ biotin, 5 mg thiamine, 10 mg CPN, 10 mg FeSO ₄ , 1 mg MnSO ₄ , 1 mg ZnSO ₄ , 200 μg CuSO ₄ and 10 mg CaCl ₂ , 2 g yeast extract 7 g casamino acid per liter Culture conditions Temperature: 30 ℃
pH: 7.0
shaking: 1,200rpm

In order to prevent depletion of glucose during the culture, when the glucose concentration was decreased to 0.5% (w / v) or less, glucose solution (90 g per 150 ml) was added. Glucose concentrations were monitored using a glucose analyzer (YSI 2700 SELECT Biochemistry Analyzer; YSI Life Science, Yellow Springs, OH, USA). Cell growth was monitored by measuring the optical density (OD) at 600 nm using a spectrophotometer (Optizen POP; Mecasys, Daejeon, Korea). FIG. 2 shows a flow diagram of a fed-batch culture of the Corynebacterium glutamicum wild-type ATCC13032 strain.

As a result, as shown in FIG. 2, a high-concentration cell culture medium having an OD ₆₀₀ = 180 level was obtained through a 33-hour fed-batch culture.

Example  2: 2 DE  ( two - dimensional 전공기 Secretory protein (Secretome) analysis

The present inventors have searched for a highly efficient protein secreted by culturing the Corynebacterium glutamicum cell cultured in Example 1 above.

To this end, 50 ml of the high-concentration cell culture obtained in Example 1 was taken and the supernatant was separated by centrifugation (5000 rpm, 15 minutes). The separated supernatant was subjected to two-dimensional electrophoresis (2DE) for proteome analysis. 2DE gel silver staining. The results are shown in Fig.

Example  3: MALDI - TOF mass 분광계  High-efficiency secretory protein screening through analysis

Protein spots that occupied the largest portion in the 2DE results (Fig. 3) of Example 2 were selected. In order to identify the protein, the gel site containing the protein spot was cut off using a knife, and the protein was isolated and degraded into a peptide form by trypsin treatment. The peptide sequences were analyzed by peptide finger printing method using MALDI (Matrix-assisted laser desorption / ionization) -TOF (time of flight) mass spectrophotometer analyzer. Based on the peptide sequence analyzed by the above method, homology analysis with the entire amino acid sequence of Corynebacterium glutamicum wild type ATCC13032 known to NCBI was performed.

As a result, the protein isolated from the 2DE gel protein spot was identified as Cgl1342 (Accession No. BA000036, SEQ ID NO: 5) protein. This protein, Cgl1342, is a hypothetical protein whose function is unknown.

Example  4: Cgl1342  Confirmation of protein secretion

In order to confirm whether the Cgl1342 protein selected in Example 3 was actually secreted at high efficiency during fermentation of Corynebacterium glutamicum ATCC13032, the expression of pCgl1342 (SEQ ID NO: 6), which is a self-promoter of Cgl1342 protein, The recombinant expression vector was constructed.

(1989), Cold Spring Harbor Laboratories) using primers of SEQ ID NO: 8 and SEQ ID NO: 9 containing a KpnI / EcoRV cleavage site using a genomic DNA derived from ATCC13032 as a template and a PCR (Sambrook et al, Molecular Cloning, Respectively. The PCR conditions were denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, and polymerization at 72 ° C for 90 seconds, and the polymerization reaction was carried out at 72 ° C for 7 minutes.

As a result, a pCgl1342_SS_Cgl1342_his_tag fragment (SEQ ID NO: 7) of about 1.1 Kb was obtained. The obtained fragment of SEQ ID NO: 7 and the pCES208 vector were treated with restriction enzyme KpnI / EcoRV and ligation was performed to prepare an expression vector of Cgl1342 protein. The recombinant expression vector prepared by the above method was named pCES208_pCgl1342_SS_Cgl1342 and transformed into Corynebacterium glutamicum ATCC13032 by the electric pulse method (Appl. Microbiol. Biothcenol. (1999) 52: 541-545) kanamycin) 25 mg / L and obtained a transformant strain and named it ATCC13032 / pCES208_pCgl1342_SS_Cgl1342.

The ATCC13032 / pCES208_pCgl1342_SS_Cgl1342 transformant strain obtained above was streaked on a BHI plate and incubated overnight at 30 ° C in order to confirm the secretion of the Cgl1342 protein in the ATCC13032 wild-type strain. The resulting mixture was incubated overnight in a 250 ml Corner-Baffle flask containing 25 ml of BHI liquid medium One platinum was inoculated and shake cultured at 30 DEG C and 200 rpm for 48 hours. The supernatant was obtained from the culture supernatant by centrifugation (5,000 rpm, 15 minutes) and 8 μl of the sample solution, which had been concentrated 5 times by acetone precipitation, was suspended in the protein loading buffer and subjected to SDS-PAGE.

As a result, it was confirmed that the Cgl1342 protein was secreted out of the cell as in the lane 1 of FIG. 4. The culture was purified using a his-tag through a Talon metal affinity resin (Clontech, Mountain View, CA) 4 in lane 2, which was confirmed by concentrated secreted protein.

These results suggest that the N-terminal region of Cgl1342, a virtual protein selected in the present invention, contains a signal peptide, which is a secretion peptide that can be used in microorganisms of the genus Corynebacterium.

Example  5: Cgl1342  signal Peptides  ( signal peptide Separation and construction of protein secretion system

N-terminal amino acid sequencing analysis of the Cgl1342 protein obtained in Example 4 in high purity was performed to confirm the amino acid sequence constituting the N-term of the Cgl1342 protein.

Thus, a signal peptide of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 1 using the Signal P software ( http://www.cbs.dtu.dk/services/SignalP/ ) It was possible to identify the signal sequence of SEQ ID NO: 3 encoding the signal peptide.

In order to confirm the increase in the expression level of the Cgl1342 signal peptide, methionine (Leucine, Leu, L) in the signal peptide of SEQ ID NO: 1 was substituted with ATG in the start codon of SEQ ID NO: , Met, M). Wherein the improved signal peptide is represented by SEQ ID NO: 2, and the improved signal sequence encoding the improved signal peptide of SEQ ID NO: 2 is represented by SEQ ID NO: The sequence comprising the nucleotide sequence upstream of the cgl1342 gene initiation codon is shown in SEQ ID NO: 38 in the signal sequence of SEQ ID NO: 3, the sequence including the nucleotide sequence preceding the cgl1342 gene initiation codon in the signal sequence of SEQ ID NO: Is shown in SEQ ID NO: 39.

The novel signal peptides developed in the present invention and the signal sequences coding the novel signal peptides are shown in Table 2 below.

Kinds order SEQ ID NO: signal
Peptides LLNRVSRIAGASAI One signal
Peptides MLNRVSRIAGASAI 2 signal
sequence TTGTTAAACAGAGTCAGTCGTATTGCAGGCGCTTCTGCAATC 3 signal
sequence ATGTTAAACAGAGTCAGTCGTATTGCAGGCGCTTCTGCAATC 4 Signal sequence ≪ 38 Signal sequence ATGTTAAACAGAGTCAGTCGTATTGCAGGCGCTTCTGCAATCACACTATGCATCGGCTTAACCACAATACTAAGCCCTACTTCCACTGCACAAAGC 39

Example  6: Xylanase  Production of Secretion Cassette and Recombinant Secretion Production Expression Vector

A protein secretion system was constructed using the signal peptide isolated and improved in Example 5 above. Thus, a signal sequence encoding the amino acid sequence of SEQ ID NO: 1 or 2 is operably linked to the downstream of the promoter sequence, and a protein secretion cassette (FIG. 1) containing a gene encoding the desired protein downstream of the sequence and a To produce a recombinant expression vector.

For this purpose, the pGap promoter constantly expressed in the Corynebacterium glutamicum wild-type ATCC13032 strain and the pCgl1342 promoter identified in the present invention were used, respectively, and xlnA, a gene of xylanase enzyme, was used as a target gene.

Specifically, PCR was performed using the primers of SEQ ID NO: 10 and SEQ ID NO: 11 using the pCES208 vector as a template (Sambrook et al., Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories). The PCR conditions were denaturation at 94 ° C for 5 minutes, followed by denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 45 seconds, and polymerization at 72 ° C for 6 minutes. The polymerisation reaction was carried out at 72 ° C for 7 minutes. As a result, a vector fragment of about 5.9 kb was obtained.

The pCES208_pCgl1342_SS_Cgl1342 vector prepared in Example 4 was used as a template as a primer of SEQ ID NO: 12 and SEQ ID NO: 13, and Streptomyces PCR was carried out using primers of SEQ ID NO: 14 and SEQ ID NO: 15 using coelicolor A3 genomic DNA as a template. The PCR conditions were denaturation at 94 ° C. for 5 minutes, denaturation at 94 ° C. for 30 seconds, annealing at 55 ° C. for 45 seconds, and polymerization at 72 ° C. for 90 seconds, and the polymerization reaction was carried out at 72 ° C. for 7 minutes. As a result, a pCgl1342 promoter_signal sequence (SEQ ID NO: 38) fragment of about 0.4 kb and an xlnA gene fragment of 1.3 kb were obtained, respectively. The vector fragments and inserts obtained by the above method were purified using a PCR purification kit (Solgent. Co), and then purified using Gibson Assembly Master Mix (NEB # 2611, Gibson, DG et al. -903), followed by ligation at 50 < [deg.] ≫ C for 1 hour to prepare a recombinant expression vector containing the pCgl1342_SS_XlnA_his_tag secretion cassette (SEQ ID NO: 16). It was named pCES208_pCgl1342_SS_XlnA vector.

In order to prepare a secretion system containing the improved signal peptide of SEQ ID NO: 2, site-direct mutagenesis PCR was performed using the pCES208_pCgl1342_SS_XlnA vector as a template and SEQ ID NO: 17 and SEQ ID NO: 18 primers to generate pCgl1342_SS1_XlnA_his_tag secretion cassette ≪ / RTI > No. 19) was constructed.

In order to confirm the possibility of protein secretion outside the microorganism by the influence of the Cgl1342 signal peptide (SEQ ID NO: 1 or SEQ ID NO: 2) alone, the pCgl1342 promoter, which is a self-promoter expressing Cgl1342 protein in Corynebacterium glutamicum ATCC13032 strain, Was replaced with the pGapA promoter known to be always expressed in the ATCC13032 strain to confirm the secretion of the protein. The same PCR as above was carried out using the pCES208_pCgl1342_SS_XlnA vector as a template and primers of SEQ ID NO: 10 and SEQ ID NO: 22 to obtain a vector fragment of about 7.4 kb. In addition, PCR was performed using primers of SEQ ID NO: 20 and SEQ ID NO: 21 using ATCC13032 genomic DNA as a template and a 0.4 kb pGapA promoter fragment was obtained. Each fragment was purified and ligated through the Gibson Assembly Master Mix method to produce a recombinant expression vector named pCES208_pGapA_SS_XlnA containing the pGapA_SS_XlnA_his_tag secretion cassette.

Example  7: Cg11342  Through the secretion system Xylanase  Secretion production confirmation

PCES208_pGapA_SS_XlnA, pCES208_pCgl1342_SS_XlnA, pCES208_pCgl1342_SS1_XlnA, and pCES208_pCgl1342_SS1_XlnA vectors were added to Corynebacterium glutamicum ATCC13032 using electric pulse method to confirm secretory production of xylanase using a protein secretion cassette containing the Cgl1342 signal peptide prepared in Example 6 (Appl. Microbiol. Biothcenol. (1999) 52: 541-545), and transformants were obtained in a selection medium containing 25 mg / L of kanamycin. . Among them, pCES208_pCgl1342_SS_XlnA was named as CA05-5009 in Corynebacterium glutamicum ATCC13032, and the corresponding strain was deposited with Korean Society of Bacterial Kidney on February 2, 2015, and it was given the deposit number of KCCM11666P. After shake culturing in the same manner as in Example 4, only the supernatant was separated to confirm secretory production of xylanase protein through SDS_PAGE.

As a result, as shown in FIG. 5-a, using the secretion cassette having the signal peptide newly isolated in the present invention (SEQ ID NO: 1), it was confirmed that the XlnA protein was secreted outside the Corynebacterium cell and identified in the culture supernatant Respectively. It was also confirmed that the amount of XlnA protein secretion was also increased in the secretion system using the modified signal peptide (SEQ ID NO: 2) in which the initiation codon was substituted with ATG in TTG.

Specifically, the absorbance at 550 nm was measured to measure the activity of xylanase enzyme.

As a result, as shown in Fig. 5-b, a pattern consistent with the secreted amount of the XlnA protein confirmed in SDS_PAGE was shown (Fig. 5-b). In addition, when the pCgl1342 promoter was replaced with the pGapA promoter, the amount of secretion relative to the pCgl1342 promoter was small. However, even when the pGapA promoter was used, the XlnA protein was secreted outside the cell and the secretion effect by the single effect of the signal peptide extracted in the present invention was confirmed.

These results suggest that the secretion peptides of SEQ ID NOS: 1 and 2, which are newly developed in the present invention, are operatively linked to various promoters regardless of the promoter derived therefrom and function as secretory peptides.

Example  8: Cgl1342  Through the secretion system Xylanase  Mass production

In order to apply the secretion production system developed in the present invention to mass production of xylanase, feed-type culture was carried out. For the mass production of xylanase, the ATCC13032 / pCES208_pCgl1342_SS1_XlnA strain which secreted the greatest amount of XlnA protein in Example 7 was used and the culturing conditions were the same as the operating conditions of the 5L fermenter performed in Example 1. [

As a result, as shown in FIG. 6-a, a high-concentration cell culture solution having a final culture time of 33 hours or OD600 = 130 or more was obtained, and the amount of XlnA protein secretion was also increased with increasing cell growth in each culture time period b). When the amount of XlnA protein secreted by the Bradford method was measured, its concentration reached a maximum of 1.06 g / L, and this value was measured by the method of Yim SS, An SJ, Kang M, Lee J, Jeong KJ. 2013. Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum . Biotechnol Bioeng 110 (11): 2959-2971.) A 40% increase over the 746 mg / L level secreted by xylenase in Corynebacterium glutamicum, the secretory production system constructed in this invention has a high concentration It was confirmed that this system is capable of mass production of target protein under cell culture conditions.

Example  9: Antibody fragment ( scFv ) Secretion cassette and recombinant expression vector production

(SS Yim et al; Appl Microbiol Biotechnol (2014) 98: 273-7) reported that secretory production was possible in Corynebacterium glutamicum ATCC13032 strain in order to confirm whether or not the secretion of enzymes other than xylanase was possible. 284), a secretion system constructed in accordance with the present invention was applied using an antibody fragment named scFv (single-chain variable fragment) as a target protein. PTac, pSod, and pGapA promoters known to be constantly expressed in Corynebacterium glutamicum in addition to the pCgl1342 promoter, in order to revalidate the effect of Cgl1342 signal peptide (SEQ ID NO: 1 or SEQ ID NO: 2) To confirm the secretion of the scFv antibody fragment by ligating the Cgl1342 signal peptide so as to be operable.

PCR was carried out using the primers of SEQ ID NO: 11 and SEQ ID NO: 24 using the pCES208_pCgl1342_SS_XlnA vector prepared in Example 6 as a template (Sambrook et al., Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories). The PCR conditions were denatured at 94 ° C for 5 minutes, followed by denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 45 seconds, and polymerization at 72 ° C for 7 minutes. The reaction was carried out at 72 ° C for 7 minutes. As a result, a vector fragment containing about 6.4 kb of the pCgl1342 promoter and the signal sequence (SEQ ID NO: 38) was obtained.

PCR was carried out using the primers of SEQ ID NO: 25 and SEQ ID NO: 26 using the pH36M2 vector (SS Yim et al; Appl Microbiol Biotechnol (2014) 98: 273-284, Respectively. The PCR conditions were denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 45 seconds, and polymerization at 72 ° C for 90 seconds, and the polymerization reaction was carried out at 72 ° C for 7 minutes. As a result, an M18 scFv gene fragment of about 0.7 kb was obtained.

The vector fragments and the M18 scFv gene fragments obtained by the above method were purified using a PCR purification kit (Solgent. Co) and then purified using a Gibson Assembly Master Mix (NEB # 2611, Gibson, DG et al. , 901-903), followed by ligation at 50 ° C for 1 hour to construct a recombinant expression vector containing the pCgl1342_SS_scFv_flag_tag secretion cassette (SEQ ID NO: 27), which was named pCES208_pCgl1342_SS_scFv vector. Site-directed mutagenesis PCR was performed using the pCES208_pCgl1342_SS_scFv vector as a template and the primers of SEQ ID NO: 17 and SEQ ID NO: 18 to construct a vector containing the improved signal peptide, thereby obtaining pCES208_pCgl1342_SS1_scFv (SEQ ID NO: 28) containing the pCGL1342_SS1_scFv_flag_tag secretion cassette Was constructed.

PCES208_pTrc_SS_scFv, pCES208_pTac_SS_scFv, pCES208_pTuf_SS_scFv, pCES208_pSod_SS_scFv, pCES208_pSod_SS_scFv, and pCES208_pSad_SS_scFv were constructed by using the above-described pCES208_pCgl1342_SS_scFv vector as a template using the primer combinations shown in Table 3 below in order to express the Cgl1342 signal sequence (SEQ ID NO: 38) , a recombinant expression vector named pCES208_pGapA_SS_scFv was constructed.

Production vector Primer used pCES208_pTrc_SS_scFv SEQ ID NO: 30 and SEQ ID NO: 31 pCES208_pTac_SS_scFv SEQ ID NO: 32 and SEQ ID NO: 33 pCES208_pTuf_SS_scFv SEQ ID NO: 34 and SEQ ID NO: 35 pCES208_pSod_SS_scFv SEQ ID NO: 36 and SEQ ID NO: 37 pCES208_pGapA_SS_scFv SEQ ID NO: 20 and SEQ ID NO: 21 pCES208_SS_scFv SEQ ID NO: 10 and SEQ ID NO: 29

The primer sequences used in the above examples are shown in Table 4 below.

Name of the primer order SEQ ID NO: pCgl1342_ss_Cgl1342 primer1_KpnI GGGTACCGGAGCCTGACTAGCGGTGTTTAAGGCACTATTATGTTGCTTTTAAACAGCATC 8 pCgl1342_ss_Cgl1342 primer2_EcoRV CGATATCTTAATGATGGTGATGGTGATGTTTTAGAGTTTTTAGTCTACCGAGATTTGTCC 9 pCES208_R ATCAAGCTTATCGATACCGT 10 pCES208_F ATCGAATTCCTGCAGCCCGG 11 pCgl1342_ss_F ACGGTATCGATAAGCTTGATAGCCTGACTAGCGGTGTTTA 12 pCgl1342_ss_R ttGGCCGGCTGGGCCTCTAGAGCTTTGTGCAGTGGAAGTA 13 xlnA_F CTACTTCCACTGCACAAAGCTCTAGAGGCCCAGCCGGCCaaggtgcgggtccagcgttggttgctgccgt 14 xlnA_R CCGGGCTGCAGGAATTCGATctattaATGATGGTGATGGTGATGGCCGAGAGCACGCTCggcgccgcggc 15 ss1_F TACATGATCAGGAGCTCTTT ATG TTAAACAGAGTCAGTCG 17 ss1_R CGACTGACTCTGTTTAA CAT AAAGAGCTCCTGATCATGTA 18 pGapA_ss_F ACGGTATCGATAAGCTTGATcatcacattggcttcaaatc 20 pGapA_ss_R CGACTGACTCTGTTTAACAAgttgtgtctcctctaaagat 21 pCES208_ss_F TTGTTAAACAGAGTCAGTCG 22 pCgl1342_ss_IST_R GCTTTGTGCAGTGGAAGTAG 24 scFv_IST_F CTACTTCCACTGCACAAAGCTCTAGAAGACATTCAGATGACCCAGACCAC 25 scFv_IST_R CCGGGCTGCAGGAATTCGATTTATCACTTATCATCGTCGTCCTTGTAGTC 26 pCES208_SS_scFv_IST_F TTGTTAAACAGAGTCAGTCG 29 pTrc_ss_F ACGGTATCGATAAGCTTGATTTGACAATTAATCATCCGGC 30 pTrc_ss_R CGACTGACTCTGTTTAACAAGGTCTGTTTCCTGTGTGAAA 31 pTac_ss_F ACGGTATCGATAAGCTTGATTCTGGCAAATATTCTGAAAT 32 pTac_ss_R CGACTGACTCTGTTTAACAAGAATTCTGTTTCCTGTGTGA 33 pTuf_ss_F ACGGTATCGATAAGCTTGATGCGTTTTAGCGTGTCAGTAG 34 pTuf_ss_R CGACTGACTCTGTTTAACAATGTATGTCCTCCTGGACTTC 35 pSod_ss_F ACGGTATCGATAAGCTTGATAAGCGCCTCATCAGCGGTAA 36 pSod_ss_R CGACTGACTCTGTTTAACAAGGGTAAAAAATCCTTTCGTA 37

Example  10: Cgl1342  Antibody fragments using secretion system ( scFv ) Secretion production confirmation

PCES208_pCgl1342_SS_scFv, pCES208_pCgl1342_SS1_scFv, pCES208_pTrc_SS_scFv, pCES208_pTac_SS_scFv, and pCES208_pTac_SS_scFv were added to Corynebacterium glutamicum ATCC13032 to confirm secretory production of the antibody fragment (scFv) using the protein secretion cassette containing the Cgl1342 signal peptide prepared in Example 9, pCES208_pTud_SS_scFv, pCES208_pSod_SS_scFv and pCES208_pGapA_SS_scFv vectors were transformed with the electric pulse method (Appl. Microbiol. Biothcenol. (1999) 52: 541-545) and then obtained transformant strains in a selection medium containing 25 mg / L of kanamycin Respectively.

After shake-culturing in the same manner as in Example 4, only the supernatant was separated and the secretion of the antibody fragment (scFv) was confirmed by ELISA (enzyme-linked immunosorbent assay) analysis.

As a result, as shown in the results of FIG. 7, it was confirmed that the antibody fragment (scFv) was also secreted out of the cell in the Corynebacterium glutamicum ATCC13032 strain using the secretion system of the present invention. In addition, although there was a difference in the secretion amount by the promoter, it was confirmed that the target protein could be secreted by the single effect by the Cgl1342 signal peptide (SEQ ID NO: 1 or SEQ ID NO: 2) identified in the present invention.

Example  11: Cgl1342  Antibody fragments using secretion system ( scFv Production of mass secretion

(HS Yim et al; Appl Microbiol Biotechnol (2014) 98: 273-7) which is a system of the signal sequence combination of the porB gene and the H36 synthetic promoter, which is the system that produced the most antibody fragment in the existing Corynebacterium glutamicum, 284) and the Cgl1342 secretion expression system of the present invention. The strains harboring the pH36M2 vector and the pCES208_pCgl1342_SS1_scFv vector were cultured in the same manner as in Example 10 to compare the amount of scFv secreted.

As a result, as shown in the result of SDS-PAGE analysis of FIG. 8A and the activity measurement of 8B, there was almost no difference in the degree of secretion of the antibody fragment (scFv) by the two secretion system through the flask culture. However, when Corynebacterium glutamicum containing the Cgl1342 secretion expression system of the present invention was subjected to oil-bed culturing and mass production of the antibody fragment was attempted, it was possible to secrete an excess of the antibody fragment as seen in FIG. This value is approximately 100 mg / L, which is more than 40% higher than the 68 mg / L of the secretion system of the H36 synthetic promoter and the porB gene signal sequence which produced the largest amount of antibody fragments in the conventional Corynebacterium glutamicum. (SS Yim et al; Appl Microbiol Biotechnol (2014) 98: 273-284). Thus, when the Cgl1342 secretion system constructed in the present invention was used, it was confirmed that mass production of target proteins of xylanase and antibody fragments was possible through high-concentration cell culture of Corynebacterium sp. Microorganism.

The above results show that the secretory peptide of SEQ ID NO: 1 and the secretory peptide of SEQ ID NO: 2, which are newly developed secretory peptides of the present invention, can be activated by secretory peptides, regardless of the promoter and target protein operatively linked thereto, Suggesting that the secretory production of the target protein is increased in the four microorganisms of the genus Bacterium. In addition, in the case of high-concentration cell culture of Corynebacterium genus microorganisms, the secretion system comprising the secretory peptides of the present invention significantly increases the secretion of the target protein and is thus effective for the mass production of the target protein It is suggestive.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Korea Microorganism Conservation Center (overseas) KCCM11666P 20150202

<110> CJ CheilJedang Corporation          Korea Advanced Institute of Science and Technology <120> KPA150069-KR <130> A novel expression cassette for secretion of protein <160> 39 <170> Kopatentin 2.0 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Cgl1342 signal peptide <400> 1 Leu Leu Asn Arg Val Ser Arg Ile Ala Gly Ala Ser Ala Ile   1 5 10 <210> 2 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Cgl1342 signal peptide (L-> M) <400> 2 Met Leu Asn Arg Val Ser Ser Ile Ala Gly Ala Ser Ala Ile   1 5 10 <210> 3 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Cgl1342 signal sequence <400> 3 ttgttaaaca gagtcagtcg tattgcaggc gcttctgcaa tc 42 <210> 4 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Cgl1342 signal sequence (TTG-> ATG) <400> 4 atgttaaaca gagtcagtcg tattgcaggc gcttctgcaa tc 42 <210> 5 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> Cgl1342 <400> 5 Leu Leu Asn Arg Val Ser Ser Ile Ala Gly Ala Ser Ala Ile Thr Leu   1 5 10 15 Cys Ile Gly Leu Thr Thr Ile Leu Ser Pro Thr Ser Thr Ala Gln Ser              20 25 30 Leu Glu Gln Ile Thr Pro Leu Pro Glu Ser Ala Ile Asp Leu Asn Ala          35 40 45 Glu Ile His Val Asn Thr Ser Asp Ile Ser Ala Glu Gln Ile Leu Gly      50 55 60 Ala Gln Asp Glu Ile Thr Thr Met Tyr Asp Ser His Asp Pro Tyr Glu  65 70 75 80 Tyr Phe Asp Thr Leu Thr Asp Ile Glu Gln Arg Ser Ile Ile Ala Ala                  85 90 95 Leu Lys Arg Asp Pro Ser Ser Leu Gln Gln Arg Gln Glu Thr Arg Leu             100 105 110 Ala Ala Gln Ser Asp Pro Tyr Lys Ile Tyr Ile Ser Gly Leu Glu Met         115 120 125 Leu Ser Cys Ile Asn Leu Val Asp Val Val Ser Cys Gly Ile Ala Asn     130 135 140 Gln Ala Ala Thr Lys Ala Asn Asn Glu Ala Val Ala Arg Tyr Pro Gly 145 150 155 160 Asp Ser Leu Arg Asn Gly Lys Gly Asp Ala Phe Arg His Cys Ser Trp                 165 170 175 Asn Ala Leu Met Thr Ile Arg Ile Gly Ser Asn Gly Ala Glu Arg Ile             180 185 190 Ala Thr Asn His Glu Thr Ile Gly Asp Gly As Ala Asp Glu Asn Ala         195 200 205 Met Asp Leu Phe Asn Asn Ala Gln Gly Arg Gln Ile Gly Ala Gly Phe     210 215 220 Ile Asn Ser Lys Asp Glu Thr Ser Ala Leu Ala Ile Cys Ala Leu Trp 225 230 235 240 Thr Asn Leu Gly Arg Leu Lys Thr Leu Lys                 245 250 <210> 6 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> Promoter of Cgl1342 <400> 6 agcctgacta gcggtgttta aggcactatt atgttgcttt taaacagcat cgaataagca 60 ctttaagcgt catttttttc catagcgaaa tacgtatttt tgagttcttt agtcatgcgc 120 cattaactag aagatctatt tcactacacc acaggcttcc ggaagttgca cattggaaaa 180 acccttagat aaatattgaa atatgaattt aataatagat tccgaacgaa atcggtgtta 240 gcgtctgtgc tgaaacaatc taatcgcgtt tcaggacacc tacatgatca ggagctcttt 300                                                                          300 <210> 7 <211> 1071 <212> DNA <213> Artificial Sequence <220> <223> pCgl1342_ss_Cgl1342_his-tag <400> 7 agcctgacta gcggtgttta aggcactatt atgttgcttt taaacagcat cgaataagca 60 ctttaagcgt catttttttc catagcgaaa tacgtatttt tgagttcttt agtcatgcgc 120 cattaactag aagatctatt tcactacacc acaggcttcc ggaagttgca cattggaaaa 180 acccttagat aaatattgaa atatgaattt aataatagat tccgaacgaa atcggtgtta 240 gcgtctgtgc tgaaacaatc taatcgcgtt tcaggacacc tacatgatca ggagctcttt 300 ttgttaaaca gagtcagtcg tattgcaggc gcttctgcaa tcacactatg catcggctta 360 accacaatac taagccctac ttccactgca caaagcctcg aacagatcac ccctttacct 420 gaatctgcaa tcgacctcaa cgccgagatt cacgtaaaca caagcgacat ttcagctgaa 480 cagatccttg gtgctcaaga tgaaatcaca actatgtacg attctcatga cccctacgag 540 tacttcgata ccctcaccga catcgaacag cgttcaataa tagcagcgct taaacgggat 600 ccgagttcac tccaacaacg ccaagaaacc cgtctcgcgg cacagtccga cccctacaaa 660 atttacatat caggcctcga aatgctttca tgcatcaatc tagttgatgt tgtatcatgc 720 gggattgcaa accaagcagc aaccaaagca aataatgagg ctgtcgcacg atacccaggc 780 gattcccttc gcaacggcaa aggcgatgca tttcggcatt gctcatggaa cgctctgatg 840 acgatacgaa tcgggagcaa tggagctgaa agaattgcaa caaaccacga gacaatcggg 900 gcggtccgg ccgatgaaaa tgcaatggac ctattcaata atgcacaagg ccgacagatc 960 ggagccggat tcattaatag taaggatgaa actagcgcgc tcgcgatatg cgcgctgtgg 1020 acaaatctcg gtagactaaa aactctaaaa catcaccatc accatcatta a 1071 <210> 8 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> pCgl1342_ss_Cgl1342 primer1_KpnI primer <400> 8 gggtaccgga gcctgactag cggtgtttaa ggcactatta tgttgctttt aaacagcatc 60                                                                           60 <210> 9 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> pCES208_R primer <400> 9 cgatatctta atgatggtga tggtgatgtt ttagagtttt tagtctaccg agatttgtcc 60                                                                           60 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pCES208_R primer <400> 10 atcaagctta tcgataccgt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pCES208_F primer <400> 11 atcgaattcc tgcagcccgg 20 <210> 12 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pCgl1342_ss_F primer <400> 12 acggtatcga taagcttgat agcctgacta gcggtgttta 40 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pCgl1342_ss_R primer <400> 13 ttggccggct gggcctctag agctttgtgc agtggaagta 40 <210> 14 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> xlnA_F primer <400> 14 ctacttccac tgcacaaagc tctagaggcc cagccggcca aggtgcgggt ccagcgttgg 60 ttgctgccgt 70 <210> 15 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> xlnA_R primer <400> 15 ccgggctgca ggaattcgat ctattaatga tggtgatggt gatggccgag agcacgctcg 60 gcgccgcggc 70 <210> 16 <211> 1749 <212> DNA <213> Artificial Sequence <220> <223> pCgl1342_ss_xlnA_his-tag <400> 16 agcctgacta gcggtgttta aggcactatt atgttgcttt taaacagcat cgaataagca 60 ctttaagcgt catttttttc catagcgaaa tacgtatttt tgagttcttt agtcatgcgc 120 cattaactag aagatctatt tcactacacc acaggcttcc ggaagttgca cattggaaaa 180 acccttagat aaatattgaa atatgaattt aataatagat tccgaacgaa atcggtgtta 240 gcgtctgtgc tgaaacaatc taatcgcgtt tcaggacacc tacatgatca ggagctcttt 300 ttgttaaaca gagtcagtcg tattgcaggc gcttctgcaa tcacactatg catcggctta 360 accacaatac taagccctac ttccactgca caaagctcta gaggcccagc cggccaaggt 420 gcgggtccag cgttggttgc tgccgttgga gcaggtgtac agctggatca gggtgccgtt 480 ggccgtgccg ttcccgacgg cgtcgaggca gaggccggac tggacgccga cgacggaccc 540 gtcggagttg aggcgccact tctggttgtc gccgccccag cagctgtaga tctggacctt 600 ggagccgttg ccggtgcctg cggcgtccag gcacttgtcg ccgtagaccc tgagctcgcc 660 cgcgtcagtg gcggcccact gctggttggt gccgctgtgg cagtcccaca gctggagctg 720 ggtgccgtcg gaggtgctgg cgtcgggcac gtcgaggcag cggcccgaac cgacgccctt 780 gatctgtccc ccgtccgcgg ggggctccga ggagtcgccg ccgttgagtg cgtcgaggac 840 ggcggtgtac gcggccttct tgctgccgtc gttgttgaac agcaacggcg tctgctccga 900 ccgccaggag tcgctgtcgc gcacacccca gcggtgatg ccgaggcagc gcgagacggc 960 caggcagtcg ttggtcacgt tggcgtaggt cgaggccggg gcgccctgga tgtccagctc 1020 ggtgatggcc acgtcgacgc cgagggcggc gaagttctgc agtgtggtgc ggaagttgct 1080 gttgtagggg ctgccgctgt tgaagtgcga ctggaagccg acgcagtcga tcggcacgcc 1140 gcgctgcttg aagtcccgca ccatgttgta catggcctgg gtcttggccc aggtccagtt 1200 ctcgacgttg tagtcgttgt agcagagctt ggcggacggg tcggcggcgc gcgcggtgcg 1260 gaaggcgacc tcgatccagt cgttgccgct gcgttgcagg ttggagtccc gccgcgctcc 1320 cgaactgccg tcggcgaagg cctcgttcac gacgtcccac tggacgatct tgcccttgta 1380 gtgggccatc acgccgttga tgtggtcgat catcgcctgg cgcagcgcgc tgccgctgag 1440 gctctgcatc cagccgggct gctgggagtg ccaggccagg gtgtggccgc gcacctgctt 1500 gccgttctgc accgcccagt tgtagacgcg gtcggcggag ctgaagttga actggccccg 1560 ctgcggttcg gtggcgtcga tcttcatctc gttctcggcc gtcaccatgt tgaactcacg 1620 gcccgcgatc gacgtgtacg tcgagtcgct cagcctgccc gaggcgatgg cggtgccgaa 1680 gtagcggccg ctctgcgccg ccgcggcgcc gagcgtgctc tcggccatca ccatcaccat 1740 cattaatag 1749 <210> 17 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> ss1_F primer <400> 17 tacatgatca ggagctcttt atgttaaaca gagtcagtcg 40 <210> 18 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> ss1_R primer <400> 18 cgactgactc tgtttaacat aaagagctcc tgatcatgta 40 <210> 19 <211> 1749 <212> DNA <213> Artificial Sequence <220> <223> pCGL1342_ss1_xlnA_his-tag <400> 19 agcctgacta gcggtgttta aggcactatt atgttgcttt taaacagcat cgaataagca 60 ctttaagcgt catttttttc catagcgaaa tacgtatttt tgagttcttt agtcatgcgc 120 cattaactag aagatctatt tcactacacc acaggcttcc ggaagttgca cattggaaaa 180 acccttagat aaatattgaa atatgaattt aataatagat tccgaacgaa atcggtgtta 240 gcgtctgtgc tgaaacaatc taatcgcgtt tcaggacacc tacatgatca ggagctcttt 300 atgttaaaca gagtcagtcg tattgcaggc gcttctgcaa tcacactatg catcggctta 360 accacaatac taagccctac ttccactgca caaagctcta gaggcccagc cggccaaggt 420 gcgggtccag cgttggttgc tgccgttgga gcaggtgtac agctggatca gggtgccgtt 480 ggccgtgccg ttcccgacgg cgtcgaggca gaggccggac tggacgccga cgacggaccc 540 gtcggagttg aggcgccact tctggttgtc gccgccccag cagctgtaga tctggacctt 600 ggagccgttg ccggtgcctg cggcgtccag gcacttgtcg ccgtagaccc tgagctcgcc 660 cgcgtcagtg gcggcccact gctggttggt gccgctgtgg cagtcccaca gctggagctg 720 ggtgccgtcg gaggtgctgg cgtcgggcac gtcgaggcag cggcccgaac cgacgccctt 780 gatctgtccc ccgtccgcgg ggggctccga ggagtcgccg ccgttgagtg cgtcgaggac 840 ggcggtgtac gcggccttct tgctgccgtc gttgttgaac agcaacggcg tctgctccga 900 ccgccaggag tcgctgtcgc gcacacccca gcggtgatg ccgaggcagc gcgagacggc 960 caggcagtcg ttggtcacgt tggcgtaggt cgaggccggg gcgccctgga tgtccagctc 1020 ggtgatggcc acgtcgacgc cgagggcggc gaagttctgc agtgtggtgc ggaagttgct 1080 gttgtagggg ctgccgctgt tgaagtgcga ctggaagccg acgcagtcga tcggcacgcc 1140 gcgctgcttg aagtcccgca ccatgttgta catggcctgg gtcttggccc aggtccagtt 1200 ctcgacgttg tagtcgttgt agcagagctt ggcggacggg tcggcggcgc gcgcggtgcg 1260 gaaggcgacc tcgatccagt cgttgccgct gcgttgcagg ttggagtccc gccgcgctcc 1320 cgaactgccg tcggcgaagg cctcgttcac gacgtcccac tggacgatct tgcccttgta 1380 gtgggccatc acgccgttga tgtggtcgat catcgcctgg cgcagcgcgc tgccgctgag 1440 gctctgcatc cagccgggct gctgggagtg ccaggccagg gtgtggccgc gcacctgctt 1500 gccgttctgc accgcccagt tgtagacgcg gtcggcggag ctgaagttga actggccccg 1560 ctgcggttcg gtggcgtcga tcttcatctc gttctcggcc gtcaccatgt tgaactcacg 1620 gcccgcgatc gacgtgtacg tcgagtcgct cagcctgccc gaggcgatgg cggtgccgaa 1680 gtagcggccg ctctgcgccg ccgcggcgcc gagcgtgctc tcggccatca ccatcaccat 1740 cattaatag 1749 <210> 20 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pGapA_ss_F primer <400> 20 acggtatcga taagcttgat catcacattg gcttcaaatc 40 <210> 21 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pGapA_ss_R primer <400> 21 cgactgactc tgtttaacaa gttgtgtctc ctctaaagat 40 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pCES208_ss_F primer <400> 22 ttgttaaaca gagtcagtcg 20 <210> 23 <211> 1876 <212> DNA <213> Artificial Sequence <220> <223> pGapA_ss_xlnA_his-tag <400> 23 catcacattg gcttcaaatc tgagacttta atttgtggat tcacgggggt gtaatgtagt 60 tcataattaa ccccattcgg gggagcagat cgtagtgcga acgatttcag gttcgttccc 120 tgcaaaaact atttagcgca agtgttggaa atgcccccgt ttggggtcaa tgtccatttt 180 tgaatgtgtc tgtatgattt tgcatctgct gcgaaatctt tgtttccccg ctaaagttga 240 ggacaggttg acacggagtt gactcgacga attatccaat gtgagtaggt ttggtgcgtg 300 agttggaaaa attcgccata ctcgcccttg ggttctgtca gctcaagaat tcttgagtga 360 ccgatgctct gattgaccta actgcttgac acattgcatt tcctacaatc tttagaggag 420 acacaacttg ttaaacagag tcagtcgtat tgcaggcgct tctgcaatca cactatgcat 480 cggcttaacc acaatactaa gccctacttc cactgcacaa agctctagag gcccagccgg 540 ccaaggtgcg ggtccagcgt tggttgctgc cgttggagca ggtgtacagc tggatcaggg 600 tgccgttggc cgtgccgttc ccgacggcgt cgaggcagag gccggactgg acgccgacga 660 cggacccgtc ggagttgagg cgccacttct ggttgtcgcc gccccagcag ctgtagatct 720 ggaccttgga gccgttgccg gtgcctgcgg cgtccaggca cttgtcgccg tagaccctga 780 gctcgcccgc gtcagtggcg gcccactgct ggttggtgcc gctgtggcag tcccacagct 840 ggagctgggt gccgtcggag gtgctggcgt cgggcacgtc gaggcagcgg cccgaaccga 900 cgcccttgat ctgtcccccg tccgcggggg gctccgagga gtcgccgccg ttgagtgcgt 960 cgaggacggc ggtgtacgcg gccttcttgc tgccgtcgtt gttgaacagc aacggcgtct 1020 gctccgaccg ccaggagtcg ctgtcgcgca caccccagac ggtgatgccg aggcagcgcg 1080 agacggccag gcagtcgttg gtcacgttgg cgtaggtcga ggccggggcg ccctggatgt 1140 ccagctcggt gatggccacg tcgacgccga gggcggcgaa gttctgcagt gtggtgcgga 1200 agttgctgtt gtaggggctg ccgctgttga agtgcgactg gaagccgacg cagtcgatcg 1260 gcacgccgcg ctgcttgaag tcccgcacca tgttgtacat ggcctgggtc ttggcccagg 1320 tccagttctc gacgttgtag tcgttgtagc agagcttggc ggacgggtcg gcggcgcgcg 1380 cggtgcggaa ggcgacctcg atccagtcgt tgccgctgcg ttgcaggttg gagtcccgcc 1440 gcgctcccga actgccgtcg gcgaaggcct cgttcacgac gtcccactgg acgatcttgc 1500 ccttgtagtg ggccatcacg ccgttgatgt ggtcgatcat cgcctggcgc agcgcgctgc 1560 cgctgaggct ctgcatccag ccgggctgct gggagtgcca ggccagggtg tggccgcgca 1620 cctgcttgcc gttctgcacc gcccagttgt agacgcggtc ggcggagctg aagttgaact 1680 ggccccgctg cggttcggtg gcgtcgatct tcatctcgtt ctcggccgtc accatgttga 1740 actcacggcc cgcgatcgac gtgtacgtcg agtcgctcag cctgcccgag gcgatggcgg 1800 tgccgaagta gcggccgctc tgcgccgccg cggcgccgag cgtgctctcg gccatcacca 1860 tcaccatcat taatag 1876 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pCgl1342_ss_IST_R primer <400> 24 gctttgtgca gtggaagtag 20 <210> 25 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> scFv_IST_F primer <400> 25 ctacttccac tgcacaaagc tctagaagac attcagatga cccagaccac 50 <210> 26 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> scFv_IST_R primer <400> 26 ccgggctgca ggaattcgat ttatcactta tcatcgtcgt ccttgtagtc 50 <210> 27 <211> 1174 <212> DNA <213> Artificial Sequence <220> <223> pCgl1342_ss_scFv_flag-tag <400> 27 agcctgacta gcggtgttta aggcactatt atgttgcttt taaacagcat cgaataagca 60 ctttaagcgt catttttttc catagcgaaa tacgtatttt tgagttcttt agtcatgcgc 120 cattaactag aagatctatt tcactacacc acaggcttcc ggaagttgca cattggaaaa 180 acccttagat aaatattgaa atatgaattt aataatagat tccgaacgaa atcggtgtta 240 gcgtctgtgc tgaaacaatc taatcgcgtt tcaggacacc tacatgatca ggagctcttt 300 ttgttaaaca gagtcagtcg tattgcaggc gcttctgcaa tcacactatg catcggctta 360 accacaatac taagccctac ttccactgca caaagctcta gaagacattc agatgaccca 420 gaccacctct tccctctccg cctccctcgg cgaccgtgtt accgtttctt gccgtgcttc 480 ccaggacatt cgcaactacc tgaactggta ccagcagaag ccagacggta ccgtcaaatt 540 ccttatctac tacacctccc gtttgcagcc tggtgttcca tcccgtttct ctggctccgg 600 ttctggcacc gattactccc tgaccatcaa caacctcgaa caggaggaca ttggtaccta 660 cttctgccag cagggcaaca ccccaccttg gaccttcggc ggtggcacca agctcgaaat 720 caaacgtggt ggtggtggtt ctgacggtgg tggttccggt ggtggcggtt ctggcggtgg 780 cggttccgaa gtgcagctgc agcagtccgg tcctgagctc gttaagccgg gcgcatccgt 840 gaagatttct tgtaaagatt ccggctacgc tttcaactcc tcttggatga actgggtcaa 900 gcagcgccct ggtcagggcc ttgaatggat cggccgtatc taccctggcg atggcgactc 960 caactacaac ggcaagttcg agggcaaagc aatcctgacc gctgataagt cctcttccac 1020 cgcctacatg cagctttctt ccttgacctc cgtggactct gcagtctact tctgcgcacg 1080 ttccggtctg ctccgttacg ctatggatta ctggggtcag ggcacctccg ttaccgtgtc 1140 ttccgactac aaggacgacg atgataagtg ataa 1174 <210> 28 <211> 1174 <212> DNA <213> Artificial Sequence <220> <223> pCgl1342_ss1_scFv_flag-tag <400> 28 agcctgacta gcggtgttta aggcactatt atgttgcttt taaacagcat cgaataagca 60 ctttaagcgt catttttttc catagcgaaa tacgtatttt tgagttcttt agtcatgcgc 120 cattaactag aagatctatt tcactacacc acaggcttcc ggaagttgca cattggaaaa 180 acccttagat aaatattgaa atatgaattt aataatagat tccgaacgaa atcggtgtta 240 gcgtctgtgc tgaaacaatc taatcgcgtt tcaggacacc tacatgatca ggagctcttt 300 atgttaaaca gagtcagtcg tattgcaggc gcttctgcaa tcacactatg catcggctta 360 accacaatac taagccctac ttccactgca caaagctcta gaagacattc agatgaccca 420 gaccacctct tccctctccg cctccctcgg cgaccgtgtt accgtttctt gccgtgcttc 480 ccaggacatt cgcaactacc tgaactggta ccagcagaag ccagacggta ccgtcaaatt 540 ccttatctac tacacctccc gtttgcagcc tggtgttcca tcccgtttct ctggctccgg 600 ttctggcacc gattactccc tgaccatcaa caacctcgaa caggaggaca ttggtaccta 660 cttctgccag cagggcaaca ccccaccttg gaccttcggc ggtggcacca agctcgaaat 720 caaacgtggt ggtggtggtt ctgacggtgg tggttccggt ggtggcggtt ctggcggtgg 780 cggttccgaa gtgcagctgc agcagtccgg tcctgagctc gttaagccgg gcgcatccgt 840 gaagatttct tgtaaagatt ccggctacgc tttcaactcc tcttggatga actgggtcaa 900 gcagcgccct ggtcagggcc ttgaatggat cggccgtatc taccctggcg atggcgactc 960 caactacaac ggcaagttcg agggcaaagc aatcctgacc gctgataagt cctcttccac 1020 cgcctacatg cagctttctt ccttgacctc cgtggactct gcagtctact tctgcgcacg 1080 ttccggtctg ctccgttacg ctatggatta ctggggtcag ggcacctccg ttaccgtgtc 1140 ttccgactac aaggacgacg atgataagtg ataa 1174 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pCES208_SS_scFv_IST_F primer <400> 29 ttgttaaaca gagtcagtcg 20 <210> 30 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pTrc_ss_F primer <400> 30 acggtatcga taagcttgat ttgacaatta atcatccggc 40 <210> 31 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pTrc_ss_R primer <400> 31 cgactgactc tgtttaacaa ggtctgtttc ctgtgtgaaa 40 <210> 32 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pTac_ss_F primer <400> 32 acggtatcga taagcttgat tctggcaaat attctgaaat 40 <210> 33 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pTac_ss_R primer <400> 33 cgactgactc tgtttaacaa gaattctgtt tcctgtgtga 40 <210> 34 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pTuf_ss_F primer <400> 34 acggtatcga taagcttgat gcgttttagc gtgtcagtag 40 <210> 35 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pTuf_ss_R primer <400> 35 cgactgactc tgtttaacaa tgtatgtcct cctggacttc 40 <210> 36 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pSod_ss_F primer <400> 36 acggtatcga taagcttgat aagcgcctca tcagcggtaa 40 <210> 37 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pSod_ss_R primer <400> 37 cgactgactc tgtttaacaa gggtaaaaaa tcctttcgta 40 <210> 38 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> Cgl1342 signal sequence <400> 38 ttgttaaaca gagtcagtcg tattgcaggc gcttctgcaa tcacactatg catcggctta 60 accacaatac taagccctac ttccactgca caaagc 96 <210> 39 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> Cgl1342 signal sequence (TTG-> ATG) <400> 39 atgttaaaca gagtcagtcg tattgcaggc gcttctgcaa tcacactatg catcggctta 60 accacaatac taagccctac ttccactgca caaagc 96

Claims (8)

(i) a promoter;
(ii) a base sequence encoding a signal peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2; And
(iii) an expression cassette expressing a target protein, which comprises a gene encoding a target protein.
2. The cassette of claim 1, wherein the nucleotide sequence encoding the signal peptide is a nucleic acid molecule of SEQ ID NO: 3 or SEQ ID NO: 4.
delete delete A vector comprising a nucleic acid molecule encoding an expression cassette of claim 1 or 2 or a signal peptide consisting of an amino acid sequence as set forth in SEQ ID NO: 1 or 2.
(i) a promoter; (ii) a base sequence encoding a signal peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2; And (iii) a cassette expressing the desired protein comprising the gene encoding the target protein, or a microorganism belonging to the genus Corynebacterium into which the vector of the fifth aspect is introduced.
The microorganism of claim 6, wherein the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum.
Culturing the microorganism of genus Corynebacterium according to claim 6 in a medium to produce a secreted protein of interest, and recovering the secreted protein from the microorganism or medium.

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