KR20190037481A - Method for Displaying Target Protein at Cell Surface Using Cell fixation Motif from Corynebacteria - Google Patents

Abstract

The present invention relates to a cell surface expression method of a target protein, which comprises a step of culturing a strain transformed with a nucleic acid encoding a fusion protein for cell surface expression, to which a cell fixation motif and the target protein are linked. According to the present invention, the target gene can be expressed more efficiently on a cell surface than a conventional gene expression system used for surface protein expression in a strain of Corynebacterium sp., and the target gene can be produced with high activity in Corynebacterium sp.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for expressing a target protein on a cell surface using a corynebacterium-derived cell-

The present invention relates to a cell surface expression method for a target protein using a novel cell-fixed host derived from Corynebacteria. More particularly, the present invention relates to a method for expressing a target protein on a cell surface using a nucleic acid encoding a fusion protein for cell surface expression, And culturing the transformed strain. The present invention also relates to a cell surface expression method of a target protein.

Corynebacterium glutamicum strains are industrially most used for amino acid production and are also used for nucleic acid and vitamin production. In addition, since proteins can be produced through cell secretion, they are actively studied in various fields, and the most widely used field is amino acid production. Corynebacterium glutamicum was found as a strain capable of producing glutamic acid at a high efficiency in the mid 1950s and was industrialized to produce various amino acids such as glutamic acid, phenylalanine, threonine, glutamine, leucine, isoleucine and histidine (Becke et al., Current Opinion in Biotechnology 23.4: 631, 2012.).

From this industrial point of view, a system for effectively decomposing polysaccharide starch or xylan is required, in which the system used expresses amylase and xylanase on the cell surface. A method has been attempted in which proteins are produced on the cell walls to decompose external polysaccharides and produce high value-added products through the cells. It also produces industrial enzymes using cell surface expression systems. This is because the enzyme can be easily recovered and recycled using the cell as a support, and the activity of the enzyme can be maintained at a high temperature.

For effective cell surface expression systems, it is important to use efficient cell immobilization matrices. This is because the target protein expression efficiency and cell stability depend on the cell immobilization matrix. There are few cases in which a cell immobilization matrix has been published in Corynebacterium spp. Corynebacterium has a different structure from that of the existing Gram-positive bacteria. Common Gram-positive bacteria have an extracellular membrane composed of peptidoglycan. Corynebacterium, on the other hand, has an extracellular membrane composed of mycolic acid outside the peptidoglycan (Marchand, Christophe H., et al., Journal of bacteriology 194.3: 587, 2012). Therefore, it is not possible to use the previously known cell-immobilized host, and a new cell-immobilized host is required to pass the most foreign micocalan and express the target protein on the cell surface.

Thus, in the present invention, as a result of efforts to develop a high-efficiency cell surface protein expression system in a strain of the genus Corynebacterium, the present inventors have found that the protein present in the extracellular membrane of the Corynebacterium glutamicum strain can be most efficiently expressed on the surface Proteins were screened through a membrane passage domain analysis, and when the selected proteins, carbonic anhydrase, amylase, xylenes, etc. were expressed in the form of a fusion protein, they were confirmed to be highly efficiently expressed on the cell surface of Corynebacterium Thereby completing the present invention.

It is an object of the present invention to provide a cell surface protein expression system that can be expressed on a cell surface with high efficiency in a strain of the genus Corynebacterium.

It is another object of the present invention to provide a cell surface expression method of a target protein using the cell surface protein expression system.

In order to accomplish the above object, the present invention provides a fusion protein for cell surface expression of a target protein to which a cell-immobilized host and an objective protein are linked, the amino acid sequence of any one of SEQ ID NOS: 1 to 3.

The present invention also provides a cell surface expression strain of a nucleic acid encoding the fusion protein, a recombinant vector for cell surface expression of the target protein containing the nucleic acid, and a nucleic acid or a target protein transformed with the recombinant vector of claim 3 do.

The present invention also provides a method for expressing a cell surface of a target protein, which comprises culturing the strain.

(A) culturing a cell surface expression strain of the target protein to express a target protein on the cell surface; And (b) recovering the target protein expressed on the cell surface.

According to the present invention, a target gene can be expressed more efficiently on a cell surface than a gene expression system used for surface protein expression in a conventional strain of Corynebacterium sp., And the target gene can be expressed with high activity in Corynebacterium Can be produced.

FIG. 1 shows the results of analysis of NCgl0535 and NCgl1337 of the cell membrane proteins of Corynebacterium glutamicum for improvement of the present invention. The region underlined indicates the secretory signal sequence and the region marked in red indicates the location where O-mycoloylation is possible.
Fig. 2 shows the results of SDS-PAGE analysis of whole secreted proteins of Corynebacterium glutamicum transformed with a plasmid containing the gene sequence fused with the entire gene of NCgl0535 and NCgl1337 with the FLAG tag. 2 represents Corynebacterium glutamicum cells transformed with pH36R-NCgl0535, 3 represents Corynebacterium glutamicum cells with plasmid pCES208 without gene, 2 represents Corynebacterium glutamicum cells transformed with pH36R-NCgl0535, 3 represents Corynebacterium glutamicum cells with pH36R- Bacterium glutamicum cells. L represents a protein marker. T represents all proteins possessed by the cell, S represents a water-soluble protein possessed by the cell, I represents a non-water-soluble protein possessed by the cell, and M represents a protein present in the cell membrane.
FIG. 3 shows the results of analysis of the C-terminal direction of the NCgl0535-FLAG fusion protein and the NCgl1337-FLAG fusion protein toward the outside of the cell. (A) And the fluorescence of the cells was measured. Red represents Corynebacterium glutamicum with the empty vector (pCES208), green represents Corynebacterium glutamicum cells with the plasmid (NCG0535) cloned with NCgl0535, blue is NCgl1337 (pH36R-NCgl1337) And Corynebacterium glutamicum cells with the cloned plasmid. The horizontal axis represents the fluorescence intensity and the vertical axis represents the number of cells. (b) was incubated with cells using FLAG tag antibody with FITC, which is a fluorescent substance, and fluorescence present in the cell surface was confirmed using a multifocal microscope. (1) is an image observed with fluorescence, (2) is an image observed with light, and (3) is a superposition of two images.
Figure 4 shows a gene expression system containing only the entire NCgl1337 gene and the transmembrane domain of NCgl1337.
FIG. 5 shows the result of producing a fusion protein by linking a gene coding for carbonic anhydrase (ngCA) to a gene expression system containing NCgl1337F and NCgl1337S, wherein (a) shows the expression amount of a carbonic anhydrase fusion protein Were compared with each other by SDS-PAGE. 1 shows Corynebacterium glutamicum cells with plasmid pCES208, 2 shows Corynebacterium glutamicum cells containing pH36R-NCgl1337F-ngCA, and 3 shows Corynebacterium glutamicum cells with pH36R-NCgl1337S-ngCA Lt; / RTI > glutamicum cells. L represents a protein marker, T represents all proteins in the cell, S represents a water-soluble protein possessed by the cell, I represents a non-water-soluble protein possessed by the cell, and M represents a protein present in the cell membrane. The arrows indicate NCgl1337full-ngCA and NCgl1337short-ngCA, respectively. (b) shows the result of measuring the fluorescence of cells by culturing in combination with the cells cultured using a FLAG tag antibody with a fluorescent substance FITC. In the case of yellow, a blank vector (pCES208) without FLAG tag antibody, And cyan blue represents Corynebacterium glutamicum with a vector (pCES208) treated with FLAG tag antibody, and blue represents Corynebacterium glutamicum with pH36R-NCgl1337F-ngCA Cells, green indicates Corynebacterium glutamicum cells with pH36-porB-ngCA, and red indicates Corynebacterium glutamicum cells with pH36R-NCgl1337S-ngCA. The horizontal axis represents fluorescence intensity and the vertical axis represents the number of cells. (c) is a comparative analysis of carbonic anhydrase activity with water saturated with carbonic acid. 1 shows the activity of the cells with the empty vector, 2 shows the activity of cells with pH36-porB-ngCA, 3 shows the activity of cells with pH36R-NCgl1337F-ngCA, and 4 shows the activity of cells with pH36R-NCgl1337S-ngCA .
FIG. 6 shows the result of producing a fusion protein by linking the xyllanase gene (XlnA) to the NCgl1337F and NCgl1337S gene expression systems, wherein (a) shows the expression level of the xylalanase fusion protein by SDS-PAGE will be. 1 represents Corynebacterium glutamicum cells containing pCES208 (empty vector), 2 represents Corynebacterium glutamicum cells containing pH36R-NCgl1337F-XlnA), and No. 3 represents pH36R-NCgl1337S-XlnA Lt; / RTI > cells expressing Corynebacterium glutamicum cells. L represents a protein marker, T represents all proteins possessed by the cell, S represents a water-soluble protein possessed by the cell, I represents a water-insoluble protein possessed by the cell, and M represents a protein present in the cell membrane. The arrows indicate NCgl1337full-XlnA and NCgl1337short-XlnA, respectively. (b) shows the result of measuring the fluorescence of cells by binding to cells cultured using a FLAG tag antibody to which FITC is attached, wherein red represents Corynebacterium glutamicum containing a vector (pCES208) Blue represents Corynebacterium glutamicum cells containing pH36R-NCgl1337F-XlnA, and green represents Corynebacterium glutamicum cells containing pH36R-NCgl1337S-XlnA. The abscissa represents the fluorescence intensity and the ordinate represents the number of corresponding cells. (c) shows the results of comparative analysis of xylanase-degrading activity of xylanase by DNS (3,5-dinitrosalicylic acid) assay.
FIG. 7 shows the result of producing a fusion protein by linking the amylase gene (Amy) to the NCgl1337F and NCgl1337S gene expression systems, wherein (a) shows the amount of expression of the amylase fusion protein by SDS-PAGE. No. 1 represents Corynebacterium glutamicum cells having pCES208, No. 2 represents Corynebacterium glutamicum cells containing pH36R-NCgl1337F-Amy and No. 3 represents Corynebacterium glume containing pH36R-NCgl1337S-Amy Tumicum cells. L represents a protein marker, T represents all proteins possessed by the cell, S represents a water-soluble protein possessed by the cell, I represents a water-insoluble protein possessed by the cell, and M represents a protein present in the cell membrane. The arrows indicate NCgl1337full-Amy and NCgl1337short-Amy, respectively. (b) shows the result of measuring the fluorescence of cells by binding to cells cultured using a FLAG tag antibody to which FITC is attached, wherein red represents Corynebacterium glutamicum containing a vector (pCES208) Blue represents Corynebacterium glutamicum cells containing pH36R-NCgl1337F-Amy, and green represents Corynebacterium glutamicum cells containing pH36R-NCgl1337S-Amy. The horizontal axis represents fluorescence intensity, and the vertical axis represents the number of corresponding cells. (c) shows the results of comparative analysis of amylase activity with EnzChek Amylase Assay Kit.

In the present invention, in order to efficiently express foreign proteins on the cell surface in Corynebacterium glutamicum, an efficient cell-fixed host was screened. In the present invention, the membrane protein existing in the extracellular membrane of the Corynebacterium glutamicum strain was analyzed by using an amino acid analysis tool to analyze the protein having the membrane passage domain. The membrane protein NCgl1337, which is a secretory protein and has an O-mycoloylation site And NCgl0535 were selected as cell-fixed hosts. Whether or not the selected two proteins are expressed in the cell membrane of Corynebacterium glutamicum was confirmed by SDS-PAGE, immunofluorescence analysis and confocal microscope scans, and the cell immobilized matrix and the exogenous protein (carbonic anhydrase, amylase Raze and xylenes) were expressed in the form of a fusion protein, they were found to be highly efficiently expressed on the cell surface of Corynebacterium.

Accordingly, in one aspect, the present invention relates to a fusion protein for cell surface expression of a target protein to which a target protein is linked, wherein the cell immobilization host is represented by the amino acid sequence of any one of SEQ ID NOS: 1 to 3.

In the present invention, SEQ ID NO: 1 represents the amino acid sequence of NCgl1337 (NCgl1337F), SEQ ID NO: 2 represents the sequence containing only the transmembrane domain of NCgl1337 (NCgl1337S), and SEQ ID NO: 3 represents the entire amino acid sequence of NCgl0535 .

In order to discover highly efficient cell immobilized cells, the membrane domain of the membrane proteins of Corynebacterium glutamicum and the secretory signal sequence were examined based on bioinformatics. In the present invention, for the first time, the function of membrane proteins of Corynebacterium glutamicum was confirmed as a cell immobilizing matrix. As a result, NCgl1337 and NCgl0535 are proteins that secrete two proteins and have positions capable of O-mycoloylation. Finally, since the protein size was small, the two proteins were selected as the immobilized cells after confirming the solubility of the modified protein.

In another aspect, the present invention relates to a nucleic acid encoding the fusion protein, a recombinant vector for cell surface expression of a target protein containing the nucleic acid, and a cell surface expression strain of the target protein transformed with the nucleic acid or the recombinant vector will be.

In the present invention, the strain is preferably Corynebacterium, more preferably Corynebacterium glutamicum, but is not limited thereto.

In one embodiment of the present invention, the entire secretory protein of Corynebacterium glutamicum transformed with a plasmid containing a gene sequence fused with the entire gene of NCgl1337 and NCgl0535 was analyzed by SDS-PAGE, and NCgl1337- FLAG tag fusion protein and NCgl0535-FLAG tag fusion protein were present in the cell membrane. Expression of NCgl1337-FLAG tag was also confirmed by flow cytometry and confocal microscopy (FIG. 3).

In another aspect, the present invention relates to a cell surface expression method of a target protein, which comprises culturing a recombinant strain for cell surface expression of the target protein.

In one embodiment of the present invention, the fusion protein is expressed in Corynebacterium glutamicum by linking a gene encoding carotenic anhydrase (ngCA) to a gene expression system containing NCgl1337F and NCgl1337S, and the result is analyzed by SDS-PAGE , The fusion protein was confirmed by flow cytometry and the activity of carbonic anhydrase was confirmed (Fig. 5). The results showed that NCgl1337 showed better yields on the cell membrane surface than the existing system (PorB; Tateno, T. et al., Applied microbiology and biotechnology 84.4: 2009) .

In another embodiment of the present invention, a fusion protein is expressed in Corynebacterium glutamicum by connecting a gene (XlnA) encoding xylalanase to a gene expression system containing NCgl1337F and NCgl1337S, and the result is analyzed by SDS-PAGE, The fusion protein was confirmed by flow cytometry and the cell xylanase activity was confirmed (Fig. 6). The cell surface expression efficiency was also high with the transmembrane domain alone (NCgl1337S).

In another embodiment of the present invention, a gene encoding amylase (Amy) is linked to a gene expression system containing NCgl1337F and NCgl1337S to express a fusion protein in Corynebacterium glutamicum, and the result is analyzed by SDS-PAGE, The fusion protein was confirmed by the analysis, and the amylase activity of the cell was confirmed (FIG. 7), and the cell surface expression efficiency was also high with the transmembrane domain alone (NCgl1337S).

In yet another aspect, the present invention provides a method for producing a target protein, comprising: (a) culturing a cell surface expression strain of the target protein, And (b) recovering the target protein expressed on the cell surface.

The term " vector " means a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in the appropriate host. The vector may be a plasmid, phage particle, or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms " plasmid " and " vector " are sometimes used interchangeably in the context of the present invention. However, the present invention includes other forms of vectors having functions equivalent to those known or known in the art.

In the present invention, the recombinant vector may include a plasmid vector, a bacteriophage vector, a cosmid vector, and a yeast artificial chromosome (YAC) vector. For the purpose of the present invention, a plasmid vector is preferably used. Typical plasmid vectors that can be used in the present invention include, in one aspect, (a) a cloning initiation point at which replication is efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site into which the foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA.

The vectors used in the present invention include a multicloning site (MCS) containing several restriction enzyme cleavage sites, which are located in the lacZ gene. Since MCS does not destroy the reading frame of the lacZ gene, lacZ is expressed in a suitable host cell to synthesize a beta-galactosidase enzyme having biological activity. When this host cell is cultured in a medium containing X-gal, a colorless chemical, X-gal is degraded by this enzyme to form an insoluble blue substance. Therefore, the host cell colonies transformed with the plasmid vector into which the foreign DNA is not inserted into the MCS are blue. Conversely, when foreign DNA is inserted into MCS, the lacZ reading frame of the plasmid vector is broken and beta-galactosidase is not produced, resulting in the formation of colorless host cell colonies.

After ligation, the vector should be transformed into the appropriate host cell. In the present invention, a preferred host cell is a prokaryotic cell. Suitable prokaryotic host cell is E.coli strain DH5a, E.coli strain JM101, E.coli K12 strain 294, strain E.coli W3110, E.coli strain X1776, E.coli XL-1Blue (Stratagene ) , and E.coli B , Correlbacterium sp., Etc., and the most preferable host cell used for producing the target protein is a Corynebacterium glutamicum.

Transformation of prokaryotic cells can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al., Supra. Alternatively, electroporation (Neumann et al., EMBO J., 1: 841 (1982)) can also be used to transform these cells.

The expression " expression control sequence " means a DNA sequence that is essential for the expression of a coding sequence operably linked to a particular host organism. Such regulatory sequences include promoters for carrying out transcription, any operator sequences for regulating such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation. For example, regulatory sequences suitable for prokaryotes include promoters, optionally operator sequences, and ribosome binding sites.

Eukaryotic cells include promoters, polyadenylation signals and enhancers. The most influential factor on the expression level of the gene in the plasmid is the promoter. As the promoter for high expression, SRα promoter and cytomegalovirus-derived promoter are preferably used.

A nucleic acid is " operably linked " when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way that the appropriate molecule (e. G., Transcriptional activator protein) is capable of gene expression when bound to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; Or a ribosome binding site is operably linked to a coding sequence if positioned to facilitate translation. Generally, " operably linked " means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be contacted. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

As used herein, the term " expression vector " is usually a recombinant carrier into which a fragment of different DNA is inserted, and generally means a fragment of double-stranded DNA. Herein, the heterologous DNA means a heterologous DNA that is not naturally found in the host cell. Once an expression vector is in a host cell, it can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (heterologous) DNA can be generated.

As is well known in the art, to increase the level of expression of a transfected gene in a host cell, the gene must be operably linked to a transcriptional and detoxification regulatory sequence that functions in the selected expression host. Preferably the expression control sequence and the gene are contained within an expression vector containing a bacterial selection marker and a replication origin. If the expression host is a eukaryotic cell, the expression vector should further comprise a useful expression marker in the eukaryotic expression host.

 As used herein, the term " transformation " means introducing DNA into a host and allowing the DNA to replicate as an extrachromosomal factor or by chromosomal integration.

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 in accordance with the gist of the present invention.

Example 1: Analysis of Corynebacterium glutamicum membrane protein by membrane passage domain analysis

It was judged that it would be suitable to use the membrane protein of a strain of genus Corynebacterium other than the other strains in order to find a highly efficient cell immobilization matrix. In addition, the membrane domain of the membrane proteins of Corynebacterium glutamicum and the secretory signal Sequences were examined based on bioinformatics. The analysis tool used the method of analyzing by inputting the amino acid sequence into http://www.cbs.dtu.dk/services/TMHMM/ and http://www.cbs.dtu.dk/services/SignalP/ . Also, since the O-mycoloylation process is required after immobilization of the membrane protein of Corynebacterium glutamicum, a specific sequence that can be identified is analyzed.

Some of the known membrane proteins have proteins whose function or mechanism is known, but some proteins are not. Therefore, in the present invention, the function of the membrane proteins of Corynebacterium glutamicum as a cell-immobilized host was first confirmed, and the results were shown (Table 1).

Protein type Amino acid number Analysis NCgl0329 315 No secretory signal sequence NCgl0776 338 No secretory signal sequence NCgl0933 126 No secretory signal sequence NCgl2375 443 No secretory signal sequence NCgl2430 183 No secretory signal sequence NCgl2779 341 No secretory signal sequence NCgl0329 315 Absence of O-mycolonylation site NCgl0336 365 Absence of O-mycolonylation site NCgl0381 319 Absence of O-mycolonylation site NCgl0513 394 Absence of O-mycolonylation site NCgl1048 278 Absence of O-mycolonylation site NCgl0987 411 More than 40 kDa NCgl1480 604 More than 40 kDa NCgl2101 483 More than 40 kDa NCgl2777 657 More than 40 kDa NCgl2789 692 More than 40 kDa NCgl0535 249 fitness NCgl1337 324 fitness

As a result, it was determined that NCgl1337 (SEQ ID NO: 1) and NCgl0535 (SEQ ID NO: 3) were suitable for the first cell immobilization host. Finally, since the protein size was small, Respectively. The results of the analysis are shown in Fig. 1, and the two proteins are secreted proteins and positions where O-mycoloylation is possible. The underlined portion of FIG. 1 represents the secretory signal sequence, and the region indicated by red represents the position where O-mycoloylation is possible.

Example 2: Analysis of NCgl1337 and NCgl0535 by SDS-PAGE, immunofluorescence, and confocal microscopy scans

To test NCG1337 and NCgl0535, the cell-immobilized candidate proteins selected in Example 1, FLAG tag (DYKDDDDK) was attached to the ends of two proteins under a strong synthetic promoter H36 and pCES208 ( J. Microbiol. Biotechnol. , 18: 639-647 , 2008) and then transformed into Corynebacterium glutamicum (ATCC13032).

Name of the primer Primer sequence NCgl0535 F
(SEQ ID NO: 10) GACCACCATATGGCGAAGAATTCTCGAATCC NCgl0535 R
(SEQ ID NO: 11) Gt; NCgl1337 F
(SEQ ID NO: 12) GTACACCATATGGCTCAGCGAAAACTGG NCgl1337R
(SEQ ID NO: 13) GTGTCAGCGGCCGCTTATTTGTCATCGTCATCTTTATAATCGGCGTTTACTCGATCTCG

The transformed Corynebacterium glutamicum was cultured in a Brain heart infusion (BHI) culture medium at 30 ^° C and 200 rpm for 24 hours. The cells were cultured in a concentration of OD ₆₀₀ of 4 by centrifugation At 4 ^° C 6000 rpm for 10 minutes, and analyzed by SDS-PAGE. SDS-PAGE was performed as follows.

SDS-PAGE

Protein expression levels in the cytoplasm, membrane, and secretosome were measured using SDS-PAGE. The cultured cells were obtained by centrifugation (Sonic, Vibra cell, Newtown, CT, USA) at 4 ° C and 6,000 rpm for 10 minutes. The obtained cells were reprecipitated in PBS (phosphate-buffered saline; 135 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.2) and separated into whole, water soluble, insoluble and membrane protein fractions. After electrophoresis on a 12% polyacrylamide gel, the gel was soaked in Coomassie brilliant blue (50% methanol, 10% acetic acid, 1 g / L Coomassie brilliant blue R-250) stained and then decolorized in destaining solution (10% methanol and 10% acetic acid)

As a result, it was confirmed that NCgl1337 and NCgl0535 were successfully expressed in the cell membrane region of the protein. The results are shown in FIG. 2. FIG. 2 shows the results of SDS-PAGE analysis of whole secreted proteins of Corynebacterium glutamicum transformed with a plasmid containing the gene sequence fused with the entire gene of NCgl0535 and NCgl1337 with the FLAG tag 1 represents Corynebacterium glutamicum cells having pCES208, which is a plasmid without any foreign gene, 2 represents Corynebacterium glutamicum cells transformed with pH36R-NCgl0535, 3 represents Corynebacterium glutamicum cells, Corynebacterium glutamicum cells transformed with pH36R-NCgl1337. L represents a protein marker. T represents all proteins possessed by the cell, S represents a water-soluble protein possessed by the cell, I represents a non-water-soluble protein possessed by the cell, and M represents a protein present in the cell membrane. Both the NCgl1337-FLAG fusion protein and the NCgl0535-FLAG fusion protein were identified in the cell membrane.

In order to confirm the degree of C-terminal orientation and the degree of efficient expression in the cell membrane region of the two proteins, immunofluorescence analysis and confocal microscope scanning were performed.

Flow-cytometric analysis of expressing cells

Transformed Corynebacterium glutamicum was sampled to a cell volume of OD ₆₀₀ 0.1, washed once and reprecipitated in PBS. The cells were immunostained with FITC (fluorescein isothiocyanate) conjugated monoclonal anti-FLAG M2 antibody (Sigma-Aldrich) for 1 hour at 4 ° C. The cells were washed once with PBS solution to remove unbound probes. The stained cells were analyzed by FACS (fluorescent activated cell sorter; MoFlo XDP, Beckman Coulter, Inc., Brea, CA, USA) to measure the fluorescence intensity caused by the immune response. As a result, as shown in FIG. 3 (a), the expression of FLAG fusion protein was confirmed in the cell membrane of Corynebacterium glutamicum (pH36R-NCgl0535) cells and Corynebacterium glutamicum (pH36R-NCgl1337) , And NCgl1337-FLAG fusion protein-expressing cells were more abundant than NCgl0535-FLAG fusion protein-expressing cells in the cell membrane.

Confocal microscopy scanning.

After the transformed Corynebacterium glutamicum was cultured, the cells were washed and reprecipitated in PBS. After immunostaining with a monoclonal anti-FLAG M2 antibody conjugated with FITC (fluorescein isothiocyanate) (Sigma-Aldrich, St. Louis, Mo., USA), the cells were placed on a glass plate coated with poly-L-lysine I left it. Cell samples were observed with a confocal microscope (LSM710, Carl Zeiss, Jena, Germany). At this time, the excitation wavelength was 488 nm argon laser, and the image was passed through only a specific light with a 505 nm filter.

As a result, it was confirmed that the direction of the C-terminal of the two proteins appeared to be in the extracellular region, and it was confirmed that NCgl1337 had fluorescence value higher than that of NCgl0535, and thus the cell membrane was more excellent. This confirms that NCgl1337 is more suitable as a cell-immobilized host (Fig. 3b).

Example 3 Construction of Cell Surface Expression System of Target Protein Using NCgl1337 Cell Immobilized Matrix

According to the results of Example 2, NCgl1337 was carried out to effectively express the target protein in the cell membrane. To do this, we constructed two systems: NCgl1337F, NCgl1337F, NCgl1337S, and O-mycoloylation. Expression was carried out under the strong promoter H36 for high efficiency production. NCgl1337F and NCgl1337S were amplified using the following forward primer NCgl1337-F, reverse primers NCgl1337F-R and NCgl1337S-R, and then introduced into restriction enzyme sites using restriction enzymes NdeI / NotI in pCES208. The prepared plasmids were named pH36R-NCgl1337F and pH36R-NCgl1337S, respectively.

Name of the primer Primer sequence NCgl1337-F
(SEQ ID NO: 14) GTACACCATATGGCTCAGCGAAAACTGG NCgl1337F-R
(SEQ ID NO: 15) TGACAGCGGCCGCTAGACACTAGTGGCGTTTACTCGATCTCG NCgl1337S-R
(SEQ ID NO: 16) TGACAGCGGCCGCTAGACACTAGTAGCCACACCACCACTTGA

Example  4: NCgl1337 Cell immobilization matrix  through Of carbonic anhydrase  Cell surface production

Carbonic anhydrase from Neisseria gonorrhoeae was expressed on the cell surface of Corynebacterium glutamicum with the system constructed in Example 3. Carbonic anhydrase is a very important enzyme for scientific and industrial purposes in preparation for global warming. Cell immobilization technology has been attempted because it can increase the stability and recovery rate of the enzyme. First PorB gene from Corynebacterium stiffness to existing widely used in cell immobilization matrix (Tateno, Toshihiro, et al Applied microbiology and biotechnology 84.4 (2009):.. 733-739) using the Neisseria gonorrhoeae (ATCC 19424) in strain A system for expressing the obtained carbonic anhydrase was prepared in pCES208. Carbonic anhydrase was amplified using CA-F and CA-R and cloned in pH36R-NCgl1337F and pH36R-NCgl1337S using SpeI / NotI restriction enzyme pH36R-NCgl1337F-CA and pH36R-NCgl1337S-CA could be obtained.

Name of the primer Primer sequence CA-F
(SEQ ID NO: 17) GGCGGTGGCGGTTCCGGCGGTGGCGGTTCCGG CA-R
(SEQ ID NO: 18) GTGTCAGCGGCCGCTTATTTGTCATCGTCATCTTTATAATCCTC

As a result, as shown in Figs. 5A and 5B, the expression level of NCgl1337 immobilized on the whole protein was higher than that of the PorB immobilized host, which is a conventional cell immobilization system. The system using only the transmembrane domain of NCgl1337 (NCgl1337S) showed similar efficiency to PorB. The activity of carbonic anhydrase was measured by hydration experiment to determine the activity value of carbonic anhydrase.

Measurement of Carbonic Anhydrase (CA) Activity by Carbon Dioxide Hydration Experiment

Carbonic anhydrase hydrates carbon dioxide and hydrogen carbonate ion (HCO ₃ ^- ) acts to lower the pH of the reaction solution. Thus, a change in pH can be detected during the hydration reaction. The activity of carbonic anhydrase is measured through the pH change at this time. A 40 mL glass bottle was covered with a rubber sputter sleeve and 12 mL of 20 mM Tris (Sigma-Aldrich) was injected. A probe from a pH meter (Thermo scientific) was injected through the hole in the tube covering the glass bottle. The pH of the Tris buffer in the vial was titrated to near pH 8.3 with 1 M hydrochloric acid (Junsei Chemical) and 400 μL of a sample of carbonic anhydrase was injected. Carbon dioxide (Special gas, Taejon, Korea) was injected into the ice flask. The pH of the solution was recorded immediately after the injection of 8 mL of carbon dioxide-saturated water. The solution was stirred magnetically at a rate of 500 rpm throughout the reaction. The activity of the carbonic anhydrase was determined as the time required for the pH to change from 8.0 to 7.0. This time data can be converted to carbonic anhydrase activity in units of WA (Wilbur-Anderson) by the following equation (I).

[수학식 I]

≪ RTI ID = 0.0 >

Where t ₀ and t represent the time required for the pH to change from 8.0 to 7.0 in a buffer-free buffer and a carbonic anhydrase-activated solution, respectively. df represents a dilution factor.

As a result of the measurement of the activity of carbonic anhydrase activity, the activity of 1.18 ± 0.19 U OD ^-1 mL ^{-1 in} the case of using the entire NCgl 1337 gene and the activity value of the existing PorB gene (0.958 ± 0.038 U OD ^-1 mL ^-1 ) % ≪ / RTI > increased activity. (0.826 ± 0.21 U OD ^-1 mL ^-1 ) similar to that of PorB when only the NCgl 1337 transmembrane domain was used (FIG. 5C). Through this, NCgl1337 shows better yield on the cell membrane surface than the existing system. This suggests that NCgl1337 exhibits better cell immobilization matrix characteristics.

Example 5: Cell surface production of xylanase via NCgl1337 cell immobilized host

With the system constructed in Example 3, the xylanase enzyme derived from Streptomyces coelicolor A3 (2) was expressed on the cell surface. A system for expressing xylenase from Streptomyces coelicolor A3 (2) (ATCC 23899) was amplified using XlnA-F and XlnA-R and cloning was carried out using pH3R-NCgl1337F and pH36R-NCgl1337S using SpeI / NotI restriction enzyme To obtain pH36R-NCgl1337F-XlnA and pH36R-NCgl1337S-XlnA.

Name of the primer Primer sequence XlnA-F
(SEQ ID NO: 19) TGACAACTAGTGCCGAGAGCACGCTCGGCGCCGCGGCGGCGCAGAGCGGTCGCTAC XlnA-R
(SEQ ID NO: 20) GCTCAGCGGCCGCTTATTTGTCATCGTCATCTTTATAATCGGTGCGGGTCCAGC

When a and b in FIG. 6 were confirmed, the fusion protein including NCgl1337S showed the highest expression level. To determine the specific activity, the following xylanase activity was measured.

Measurement of reducing sugar using 3,5-dinitrosalicylic acid (DNS)

1 mL of the cell culture solution was centrifuged at 6000 rpm for 10 minutes using a centrifuge. The precipitated cells were washed once with PBS and precipitated again. Finally, 1 mL of 0.2 M potassium phosphate buffer pH 6.3 was prepared. Xylan (Sigma) solution prepared at 2.5% concentration, 4 mL of 0.2M potassium phosphate buffer pH 6.3 and 1 mL of cells were added and reacted at 30 ^° C. Reacting 500μL of the solution was precipitated in a centrifuge for 13000rpm 10 minutes after the supernatant 300μL mix put 900μL DNS solution (1% dinitrosalicylic aicd, 0.2% phenol, 0.05% soudium sulfate, 1% sodium hydroxide), at 100 ^o C The mixture was boiled for 5 minutes and the activity was calculated by measuring the absorbance at 550 nm. 1 U of enzyme was defined as making 1 μmole of reducing sugar for 30 minutes at pH 6.3.

As a result of the activity test, cells having pH36R-NCgl1337F-XlnA had an activity of 1.03 ± 0.074 U / mL, and cells having a plasmid of pH36R-NCgl1337S-XlnA showed 4.22 ± 0.053 U / mL activity (FIG. . As a result, the cell surface expression efficiency was shown by the membrane passage domain alone.

Example 6: Cell surface production of amylase via NCgl1337 cell immobilized host

Amylase enzyme derived from Streptococcus bovis was expressed on the cell surface with the system constructed in Example 3. Amy-F and Amy-R were used to amplify the amylase-expressing system obtained from Streptococcus bovis (ATCC 33317). Cloning was carried out using pH3R-NCgl1337F and pH36R-NCgl1337S using SpeI / NotI restriction enzyme to obtain pH36R-NCgl1337F-Amy And pH36R-NCgl1337S-Amy.

Name of the primer Primer sequence Amy-F
(SEQ ID NO: 21) TGACAACTAGTGATGAACAAGTGTCAATGAAAGA Amy-R
(SEQ ID NO: 22) TGATGCGGCCGCTTATTTGTCATCGTCATCTTTATAATCTTTTAGCCCATCTTTATTATAGTTTCCAG

When a and b in FIG. 7 were confirmed, the fusion protein system having NCgl1337S showed the highest expression amount. To determine the specific activity, the following amylase activity was measured.

Measurement of amylase activity using fluorescence

1 mL of the cell culture solution was centrifuged at 6000 rpm for 10 minutes using a centrifuge. The precipitated cells were washed once with PBS and precipitated again. Finally, the precipitated cells were redissolved in 1 mL of the active solution (0.05 mM MOPS, 0.2 mM sodium azide, pH 6.9). DQ ^TM starch solution was attached to a 200 μg / mL BODIPY ^® FL dye of 100 μL, mixed with the cells verify ^{(Molecular Probes TM, Eugene, OR} , USA) the activity was reacted at room temperature for 30 minutes. After the reaction solution was precipitated at 13000 rpm for 10 minutes, the fluorescence was measured with a TECAN Infinite M200 Pro ELISA plate reader (Tecan Group Ltd., Miltenorf, Switzerland). 1 U of enzyme was defined as making 1.0 mg of reducing sugar for 30 minutes at pH 6.9.

As a result of the activity test, cells having pH36R-NCgl1337F-Amy had an activity of 3.58 ± 1.2 U / mL, and cells having a plasmid of pH36R-NCgl1337S-Amy showed activity of 50.8 ± 5.2 U / mL (FIG. . As a result, the cell surface expression efficiency was shown by the membrane passage domain alone.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea Advanced Institute of Science and Technology <120> Method for Displaying Target Protein at Cell Surface Using Cell          fixation Motif from Corynebacteria <130> P17-B225 <160> 22 <170> Kopatentin 2.0 <210> 1 <211> 324 <212> PRT <213> Artificial Sequence <220> <223> NCgl1337 full sequence <400> 1 Met Ala Gln Arg Lys Leu Ala Ser Val Ile Gly Ala Ala Leu Ala Ala   1 5 10 15 Ser Ala Val Leu Val Gly Leu Met Thr Pro Ala Thr Ala Gln Ser Ser              20 25 30 Gly Ser Ser Thr Asp Ile Thr Arg Ala Leu Thr Ser Ser Gly Gly          35 40 45 Val Ala Asp Ser Arg Ala Pro Glu Gly Gly Ala Lys Val Val Val Phe      50 55 60 Gly Asp Ser His Thr Ser Gly Thr Asn Ala Pro Phe Arg Thr Asp Glu  65 70 75 80 Arg Gly Cys Leu Lys Gly Ala Asn Asn Trp Ala Asp Gln Leu Gln Ser                  85 90 95 Gln Leu Gly Leu Gly Ala Gly Asp Leu Ile Asp Val Ser Cys Ser Gly             100 105 110 Ala Ser Ile Asn Ser Asp Gly Phe His Phe Ser Asp Glu Val Arg His         115 120 125 Ala Glu Ala Arg Gly Ala Ile Gly Pro Asn Thr Thr Asp Ile Phe Val     130 135 140 Gln Leu Gly Lys Asn Asp Gln Trp Gly Leu Ser Asn Val Asn Leu Leu 145 150 155 160 Gln Ser Val Gln Thr Cys Leu Thr Asp Val Phe Ala Gly Cys Gly Asp                 165 170 175 Ala Ala Val Ala Ala Gly Lys Met Gln Asp Pro Asn Ala Val Thr Ala             180 185 190 Glu Asn Tyr Ala Glu Arg Met Lys Pro Val Ile Asp Tyr Leu Lys Tyr         195 200 205 Tyr Ala Pro Asn Ala Glu Ile Thr Leu Val Gly Tyr Gln Glu Tyr Thr     210 215 220 Ala Arg Ser Gly Ser Gln Val Cys Val Arg Leu Gly Gly Thr Pro Leu 225 230 235 240 Val Lys Asn Asp Ala Pro Ala Leu Val Ser Phe Met Asn Lys Leu Asp                 245 250 255 Met Ala Ile Asp Gly Ala Ala Gly Ile Leu Gly Val Ser His Val Asp             260 265 270 Leu Arg Ser Ala Thr Glu Gly His Asp Ser Cys Ser Asn Asp Pro Trp         275 280 285 Val Asn Gly Val Phe Asp Ala Arg Ala Glu Ile Val Gly Gly Pro Trp     290 295 300 His Pro Ser Val Lys Gly Asp Ser Val Thr Ala Gly Ile Leu Arg Asp 305 310 315 320 Arg Val Asn Ala                 <210> 2 <211> 50 <212> PRT <213> Artificial Sequence <220> <223> NCgl1337S <400> 2 Met Ala Gln Arg Lys Leu Ala Ser Val Ile Gly Ala Ala Leu Ala Ala   1 5 10 15 Ser Ala Val Leu Val Gly Leu Met Thr Pro Ala Thr Ala Gln Ser Ser              20 25 30 Gly Ser Ser Thr Asp Ile Thr Arg Ala Leu Thr Ser Ser Gly Gly          35 40 45 Val Ala      50 <210> 3 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> NCgl0535 <400> 3 Met Ala Lys Asn Ser Arg Ile Arg Tyr Ser Ala Ser Ile Lys Arg Ala   1 5 10 15 Ala Ala Ile Leu Thr Ala Ala Thr Ser Ala Leu Ile Ala              20 25 30 Val Pro Ala Thr Ala Ser Ala Gln Asp Leu Ala Thr Gly Ser Ser Gln          35 40 45 Ile Gln Thr Asp Ala Arg Glu Gly Ala Trp Ala Thr Arg Asn Thr Ile      50 55 60 Gln Asp Gln Leu Ala Ser Ile Gly Pro Ala Ala Leu Pro Val Arg Ala  65 70 75 80 Ala Val Asp Asn Ale Ile Asn Gly Met Phe Pro Gly Leu Val Asp Glu                  85 90 95 Lys Val Ala Glu Glu Glu Ala Ala Arg Ala Glu Ala Glu Arg Glu             100 105 110 Ala Ala Ala Ala Ala Gla Ala Ala Ala Arg Ala Ala Ala Glu Glu         115 120 125 Ala Ala Arg Phe Asp Arg Gly Ser Cys Pro Ala Ile Ala Asp Val Cys     130 135 140 Val Asp Ile Asp Gly Gly Arg Thr Trp Leu Gln Glu Asn Gly Gln Val 145 150 155 160 Thr Tyr Gly Ala Val Val Ser Ser Gly Gly Val Gly Gln Glu Thr                 165 170 175 Pro Arg Gly Thr Phe Tyr Ile Asn Arg Lys Val Lys Asp Glu Ile Ser             180 185 190 Tyr Glu Phe Gly Asn Ala Pro Met Tyr Ala Met Tyr Phe Thr Tyr         195 200 205 Asn Gly His Ala Phe His Gln Gly Asn Val Ala Thr Thr Ser Ala Gly     210 215 220 Cys Val Arg Leu Asn Thr Gln Asp Ala Ile Tyr Tyr Phe Asn Asn Val 225 230 235 240 Gly Ile Gly Asp Met Val Tyr Ile Tyr                 245 <210> 4 <211> 975 <212> DNA <213> Artificial Sequence <220> <223> NCgl1337 full sequence <400> 4 atggctcagc gaaaactggc ctctgtgatc ggtgcagcat tggcagcatc tgctgtactg 60 gttggattaa tgacacccgc aacagcacaa agtagtggca gctcatcaac agacatcact 120 cgagcactca cctcaagtgg tggtgtggct gatagccgtg ctcctgaagg tggcgcaaag 180 gtcgttgttt tcggtgactc ccacacctct ggcaccaatg ctccattccg taccgatgag 240 cgtggctgcc tcaagggtgc aaacaactgg gcagatcagc tgcagtctca gctgggactt 300 ggcgcgggag acctcattga tgtctcctgc tccggtgcat cgatcaactc tgatggattc 360 cacttctctg atgaagtccg ccatgctgaa gctcgtggcg caatcggccc aaacaccacc 420 gatatttttg ttcagttggg caagaatgat cagtggggcc tttccaatgt gaaccttctg 480 cagtctgttc agacctgctt gactgatgtg ttcgctggtt gtggcgatgc tgcggttgct 540 gctggcaaga tgcaggatcc aaatgcagtt actgctgaaa actatgcaga gcgcatgaag 600 ccagtcattg actacttgaa gtactacgca ccaaacgcag agatcacctt ggttggttac 660 caggaataca ccgctcgcag cggaagtcag gtatgtgttc gccttggtgg aaccccactg 720 gtgaaaaatg atgcacctgc gctggtttcg ttcatgaaca agttggacat ggcgattgat 780 ggtgctgctg gaatcctcgg cgtcagccac gttgatctgc gtagcgcgac tgaagggcac 840 gacagctgct ccaacgatcc ttgggtcaac ggtgtctttg atgcacgtgc agaaatcgtc 900 ggcggtccgt ggcacccatc tgttaaggga gactcggtta ctgcagggat cctgcgagat 960 cgagtaaacg cctaa 975 <210> 5 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> NCgl1337S <400> 5 atggctcagc gaaaactggc ctctgtgatc ggtgcagcat tggcagcatc tgctgtactg 60 gttggattaa tgacacccgc aacagcacaa agtagtggca gctcatcaac agacatcact 120 cgagcactca cctcaagtgg tggtgtggct 150 <210> 6 <211> 750 <212> DNA <213> Artificial Sequence <220> <223> NCgl0535 <400> 6 atggcgaaga attctcgaat ccgatacagc gcgtcaatca agcgtgccgc agctgcaatc 60 ctcaccgcag ctgctacctc agtcgcgttg atcgctgtgc cagcaactgc ttcagcacag 120 gacctcgcaa ccggcagctc ccagatccag actgatgctc gtgaaggtgc gtgggcaacc 180 cgcaacacca tccaagacca acttgcctcc attgggccag cagccctccc agtccgcgca 240 gcggtagaca atgccatcaa cggcatgttc ccaggacttg ttgatgaaaa ggttgcagca 300 gagcaggaag ctgcacgcgc agaagctgag cgcgaagcag cagctgcacg tgaagcagaa 360 gcagcccgcg tagccgcaga agaagccgca cgctttgacc gcggctcttg cccagcaatc 420 gctgatgtct gcgtggacat tgatggtgga cgtacctggc tgcaggaaaa cggtcaggtc 480 acctacggtg cagtcccagt ttcctccggc ggagttggcc aggaaacccc tcgcggaacg 540 ttctacatca accgcaaggt caaggatgaa atctcttacg agttcggtaa cgccccaatg 600 ccgtacgcca tgtacttcac ctacaacggc cacgcattcc accagggcaa tgttgcgact 660 acttccgctg gttgtgttcg cctaaacact caagatgcca tctactactt caacaacgtt 720 ggcatcggcg acatggtgta catctactaa 750 <210> 7 <211> 230 <212> PRT <213> Artificial Sequence <220> <223> carbonic anhydrase <400> 7 Met Ala Met Gly His Gly Asn His Thr His Trp Gly Tyr Thr Gly His   1 5 10 15 Asp Ser Pro Glu Ser Trp Gly Asn Leu Ser Glu Glu Phe Arg Leu Cys              20 25 30 Ser Thr Gly Lys Asn Gln Ser Pro Val Asn Ile Thr Glu Thr Val Ser          35 40 45 Gly Lys Leu Pro Ala Ile Lys Val Asn Tyr Lys Pro Ser Met Val Asp      50 55 60 Val Glu Asn Asn Gly His Thr Ile Gln Val Asn Tyr Pro Glu Gly Gly  65 70 75 80 Asn Thr Leu Thr Val Asn Gly Arg Thr Tyr Thr Leu Lys Gln Phe His                  85 90 95 Phe His Val Ser Ser Glu Asn Gln Ile Lys Gly Arg Thr Phe Pro Met             100 105 110 Glu Ala His Phe Val His Leu Asp Glu Asn Lys Gln Pro Leu Val Leu         115 120 125 Ala Val Leu Tyr Glu Ala Gly Lys Thr Asn Gly Arg Leu Ser Ser Ile     130 135 140 Trp Asn Val Met Met Thr Ala Gly Lys Val Lys Leu Asn Gln Pro 145 150 155 160 Phe Asp Ala Ser Thr Leu Leu Pro Lys Arg Leu Lys Tyr Tyr Arg Phe                 165 170 175 Ala Gly Ser Leu Thr Thr Pro Pro Cys Thr Glu Gly Val Ser Trp Leu             180 185 190 Val Leu Lys Thr Tyr Asp His Ile Asp Gln Ala Gln Ala Glu Lys Phe         195 200 205 Thr Arg Ala Val Gly Ser Glu Asn Asn Arg Pro Val Gln Pro Leu Asn     210 215 220 Ala Arg Val Val Ile Glu 225 230 <210> 8 <211> 436 <212> PRT <213> Artificial Sequence <220> <223> xylanase <400> 8 Ala Glu Ser Thr Leu Gly Ala Ala Ala Gln Ser Gly Arg Tyr Phe   1 5 10 15 Gly Thr Ala Ile Ala Ser Gly Arg Leu Ser Asp Ser Thr Tyr Thr Ser              20 25 30 Ile Ala Gly Arg Glu Phe Asn Met Val Thr Ala Glu Asn Glu Met Lys          35 40 45 Ile Asp Ala Thr Glu Pro Gln Arg Gly Gln Phe Asn Phe Ser Ser Ala      50 55 60 Asp Arg Val Tyr Asn Trp Ala Val Gln Asn Gly Lys Gln Val Arg Gly  65 70 75 80 His Thr Leu Ala Trp His Ser Gln Gln Pro Gly Trp Met Gln Ser Leu                  85 90 95 Ser Gly Ser Ala Leu Arg Gln Ala Met Ile Asp His Ile Asn Gly Val             100 105 110 Met Ala His Tyr Lys Gly Lys Ile Val Gln Trp Asp Val Val Asn Glu         115 120 125 Ala Phe Ala Asp Gly Ser Ser Gly Ala Arg Arg Asp Ser Asn Leu Gln     130 135 140 Arg Ser Gly Asn Asp Trp Ile Glu Val Ala Phe Arg Thr Ala Arg Ala 145 150 155 160 Ala Asp Pro Ser Ala Lys Leu Cys Tyr Asn Asp Tyr Asn Val Glu Asn                 165 170 175 Trp Thr Trp Ala Lys Thr Gln Ala Met Tyr Asn Met Val Arg Asp Phe             180 185 190 Lys Gln Arg Gly Val Pro Ile Asp Cys Val Gly Phe Gln Ser His Phe         195 200 205 Asn Ser Gly Ser Pro Tyr Asn Ser Asn Phe Arg Thr Thr Leu Gln Asn     210 215 220 Phe Ala Ala Leu Gly Val Asp Val Ala Ile Thr Glu Leu Asp Ile Gln 225 230 235 240 Gly Ala Pro Ala Ser Thr Tyr Ala Asn Val Thr Asn Asp Cys Leu Ala                 245 250 255 Val Ser Arg Cys Leu Gly Ile Thr Val Trp Gly Val Arg Asp Ser Asp             260 265 270 Ser Trp Arg Ser Glu Gln Thr Pro Leu Leu Phe Asn Asn Asp Gly Ser         275 280 285 Lys Lys Ala Ala Tyr Thr Ala Val Leu Asp Ala Leu Asn Gly Gly Asp     290 295 300 Ser Ser Glu Pro Pro Ala Asp Gly Gly Gln Ile Lys Gly Val Gly Ser 305 310 315 320 Gly Arg Cys Leu Asp Val Pro Asp Ala Ser Thr Ser Asp Gly Thr Gln                 325 330 335 Leu Gln Leu Trp Asp Cys His Ser Gly Thr Asn Gln Gln Trp Ala Ala             340 345 350 Thr Asp Ala Gly Glu Leu Arg Val Tyr Gly Asp Lys Cys Leu Asp Ala         355 360 365 Ala Gly Thr Gly Asn Gly Ser Lys Val Gln Ile Tyr Ser Cys Trp Gly     370 375 380 Gly Asp Asn Gln Lys Trp Arg Leu Asn Ser Asp Gly Ser Val Val Gly 385 390 395 400 Val Gln Ser Gly Leu Cys Leu Asp Ala Val Gly Asn Gly Thr Ala Asn                 405 410 415 Gly Thr Leu Ile Gln Leu Tyr Thr Cys Ser Asn Gly Ser Asn Gln Arg             420 425 430 Trp Thr Arg Thr         435 <210> 9 <211> 703 <212> PRT <213> Artificial Sequence <220> <223> amylase <400> 9 Asp Glu Gln Val Ser Met Lys Asp Gly Thr Ile Leu His Ala Trp Cys   1 5 10 15 Trp Ser Phe Asn Thr Ile Lys Asp Asn Met Gln Ala Ile Lys Asp Ala              20 25 30 Gly Tyr Thr Ser Val Gln Thr Ser Pro Ile Asn Thr Val Val Ala Gly          35 40 45 Glu Gly Gly Asn Lys Ser Leu Lys Asn Trp Tyr Tyr Gln Tyr Gln Pro      50 55 60 Thr Ile Tyr Lys Ile Gly Asn Tyr Gln Leu Gly Thr Glu Glu Glu Phe  65 70 75 80 Lys Glu Met Asn Arg Val Ala Asp Gln Tyr Gly Ile Lys Ile Ile Val                  85 90 95 Asp Ala Val Leu Asn His Thr Thr Ser Asp Tyr Asn Gln Ile Ser Gln             100 105 110 Glu Ile Lys Asn Ile Pro Asn Trp Thr His Gly Asn Thr Leu Ile Ser         115 120 125 Asp Trp His Asn Arg Tyr Asp Val Thr Gln Asn Ala Leu Leu Thr Leu     130 135 140 Tyr Asp Trp Asn Thr Gln Asn Glu Tyr Val Gln Gln Tyr Leu Leu Ser 145 150 155 160 Tyr Leu Lys Gln Ala Val Ala Asp Gly Ala Asp Gly Phe Arg Tyr Asp                 165 170 175 Ala Ala Lys His Ile Glu Leu Pro Gly Glu Tyr Gly Ser Asn Phe Trp             180 185 190 Asn Val Ile Leu Asn Asn Gly Ser Glu Phe Gln Tyr Gly Glu Ile Leu         195 200 205 Gln Asp Asp Val Ser Asn Asp Ala Gly Tyr Gly Lys Leu Met Ser Ile     210 215 220 Thr Ala Ser Asn Tyr Gly Gln Lys Ile Arg Ser Ala Leu Lys Asp Arg 225 230 235 240 His Ile Ser Ala Gly Asn Leu Met Asn Tyr Gln Val Ser Gly Val Asp                 245 250 255 Ala Ala Asn Leu Val Thr Trp Val Glu Ser His Asp Asn Tyr Ala Asn             260 265 270 Asp Asp Gln Glu Ser Thr Trp Met Asn Asp Ser Asp Ile Gly Leu Gly         275 280 285 Trp Ala Met Ile Thr Ala Arg Ala Lys Gly Thr Pro Leu Phe Phe Ser     290 295 300 Arg Pro Val Gly Gly Gly Asn Gly Thr Arg Phe Pro Gly Gln Ser Gln 305 310 315 320 Ile Gly Asp Ala Gly Ser Asn Leu Tyr Lys Asp Ala Thr Val Thr Ala                 325 330 335 Val Asn Lys Phe His Asn Ala Met Val Gly Glu Ser Glu Tyr Leu Arg             340 345 350 Asn Pro Gly Gly Asp Glu Gln Val Ala Met Ile Glu Arg Gly Thr Lys         355 360 365 Gly Ala Val Ile Val Asn Leu Val Asp Gly Asp Lys Gln Ile Asn Ser     370 375 380 Glu Thr Asn Leu Ala Asp Gly Thr Tyr Thr Asp Lys Val Ser Gly Arg 385 390 395 400 Gln Phe Asn Val Ser Asn Gly Arg Ile Thr Gly Ser Val Pro Ser Arg                 405 410 415 Ser Ala Val Val Leu Tyr Asp Asp Gln Ala Ser Gln Ala Ala Gln Val             420 425 430 Ser Val Asp Gly Tyr Lys Glu Gly Asp Asn Ser Ile Ser Lys Ala Thr         435 440 445 Glu Val Thr Leu Lys Ala Lys Asn Ala Asp Ser Ala Thr Tyr Lys Leu     450 455 460 Gly Asn Gly Gln Glu Val Ala Tyr Lys Asp Gly Asp Lys Val Thr Val 465 470 475 480 Gly Glu Gly Leu Glu Ala Gly Gln Ser Thr Thr Leu Thr Leu Thr Ala                 485 490 495 Thr Gly Ala Asp Gly Gln Ser Thr Thr Lys Thr Tyr Thr Phe Thr Met             500 505 510 Lys Asp Pro Ser Ala Glu Thr Asn Ile Tyr Phe Gln Asn Pro Asp Asn         515 520 525 Trp Ser Glu Val Tyr Ala Tyr Met Tyr Ser Ala Lys Asp Asn Lys Leu     530 535 540 Leu Gly Ala Trp Pro Gly Thr Lys Met Thr Lys Glu Ala Ser Gly Arg 545 550 555 560 Tyr Ser Ile Thr Val Ala Ser Tyr Ala Glu Glu Gly Val Lys Val                 565 570 575 Ile Phe Thr Asn Asn Gln Gly Ser Gln Tyr Pro Gln Asn Glu Gly Phe             580 585 590 Asp Phe Lys Ala Glu Gly Leu Tyr Ser Lys Ala Gly Leu Met Pro Asp         595 600 605 Val Pro Ala Gly Lys Thr Arg Val Thr Phe Asp Asn Pro Gly Gly Trp     610 615 620 Asp Ser Ala Asn Ala Tyr Leu Tyr Tyr Gly Asn Pro Val Gln Tyr Pro 625 630 635 640 Leu Gly Val Trp Pro Gly Thr Gln Met Thr Lys Asp Asp Ala Gly Asn                 645 650 655 Phe Tyr Leu Asp Leu Pro Glu Glu Tyr Ala Asp Val Asn Ala Lys Ile             660 665 670 Ile Phe Asn Gln Pro Gly Thr Ser Asn Gln Tyr Pro Tyr Ser Glu Gly         675 680 685 Phe Asn Leu Val Lys Ser Gly Asn Tyr Asn Lys Asp Gly Leu Lys     690 695 700 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gaccaccata tggcgaagaa ttctcgaatc c 31 <210> 11 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gtcagcggcc gcttatttgt catcgtcatc tttataatcg tagatgtaca ccatgtcgc 59 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gtacaccata tggctcagcg aaaactgg 28 <210> 13 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gtgtcagcgg ccgcttattt gtcatcgtca tctttataat cggcgtttac tcgatctcg 59 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gtacaccata tggctcagcg aaaactgg 28 <210> 15 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 tgacagcggc cgctagacac tagtggcgtt tactcgatct cg 42 <210> 16 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 tgacagcggc cgctagacac tagtagccac accaccactt ga 42 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ggcggtggcg gttccggcgg tggcggttcc gg 32 <210> 18 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gtgtcagcgg ccgcttattt gtcatcgtca tctttataat cctc 44 <210> 19 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 tgacaactag tgccgagagc acgctcggcg ccgcggcggc gcagagcggt cgctac 56 <210> 20 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gctcagcggc cgcttatttg tcatcgtcat ctttataatc ggtgcgggtc cagc 54 <210> 21 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 tgacaactag tgatgaacaa gtgtcaatga aaga 34 <210> 22 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 tgatgcggcc gcttatttgt catcgtcatc tttataatct tttagcccat ctttattata 60 gtttccag 68

Claims (8)

A fusion protein for cell surface expression in which a cell-immobilized host represented by an amino acid sequence of any one of SEQ ID NOS: 1 to 3 is linked to a target protein.
A nucleic acid encoding the fusion protein of claim 1.
A recombinant vector for expression of a cell surface of a target protein containing the nucleic acid of claim 2.
A cell-surface expression strain of the nucleic acid of claim 2 or the target protein transformed with the recombinant vector of claim 3.
5. The method according to claim 4, wherein the strain is Corynebacterium,
6. The strain according to claim 5, wherein the strain is Corynebacterium glutamicum.
A cell surface expression method of a target protein, which comprises culturing the strain of claim 4.
A method for producing a target protein comprising the steps of:
(a) culturing the strain of claim 4 to express the desired protein on the cell surface; And
(b) recovering the target protein expressed on the cell surface.

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