KR101837636B1 - Secretory production of L-asparaginase in recombinant Corynebacterium - Google Patents

Abstract

The present invention relates to a method for expressing and secreting an Escherichia coli or an Earnia-derived asparaginase corresponding to a heterologous protein using a recombinant strain of Corynebacterium, and a method of secretory signal sequence binding to the N-terminal of asparaginase It is possible to recover asparaginase which has not undergone transformation from the recombinant Corynebacterium in high purity.

Description

[0002] Secretory production of L-asparaginase in recombinant Corynebacterium using a recombinant strain of Corynebacterium [

The present invention is a recombinant vector and method for producing and purifying asparaginase, a heterologous protein, with high yield using Corynebacterium .

Asparaginase is a protein that is used for various purposes. Typically, Esparichinase E. coli or Erwinia carotovora . Uses of asparaginase include, for example, a therapeutic agent for acute lymphoblastic leukemia, inhibition of the formation of acrylamide, which is a carcinogenic substance, and the like.

For the production of such industrially useful asparaginase, various studies have been carried out. In general, a method of transforming Escherichia coli, which is an asparaginase producing strain, with a recombinant vector to which various procedures are applied to increase the expression efficiency of asparaginase are common. For example, Korean Patent No. 1408062 B1 also discloses a method for producing asparaginase from E. coli transformed with a recombinant vector to which an arbitrary manipulation is applied. The asparaginases of high purity can be obtained by controlling the culture conditions of Escherichia coli or Earl's Wii strains, and optimizing the process of recovering and purifying proteins from the strains after culturing. However, even if asparaginase can be obtained at a high yield, a complicated process of purifying the asparaginase from these strains is required, which may cause problems such as side reactions during the purification process. In addition, there is also a problem that a complicated purification process takes a lot of time and costs.

Korea registered patent KR 1408062 B1

The present invention aims at solving the problem of production and purification of asparaginase using an asparaginase, which is an exogenous protein, by using Corynebacterium, and using an asparaginase-derived strain such as Escherichia coli.

The present invention provides recombinant vectors for asparaginase expression and secretion enhancement, including the asparaginase gene associated with a signal sequence derived from Corynebacterium .

The present invention provides a Corynebacterium (Corynebacterium) transfected a Corynebacterium recombinant strain converted to a recombinant vector for, asparaginase expression and secretion increased containing asparaginase (asparaginase) gene in combination with the derived signal sequence .

The present invention relates to a method for producing asparaginase, which comprises transforming a Corynebacterium- derived signal sequence with an asparaginase gene, a Corynebacterium transformed with a recombinant vector for expression and secretion of asparaginase, Provides a method of production.

It is an object of the present invention to provide a method for producing asparaginase which is efficiently expressed and secreted as an exogenous protein in Corynebacterium and which is capable of recovering asparaginase with high efficiency through a simple purification process, It is safe from a variety of sources.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing the construction of a plasmid used for functional analysis of signal sequences usable in Corynebacterium glutamicum.
FIG. 2 shows the results of comparing the expression efficiency of GFPuv by transforming each of the 14 recombinant strains with the GFPuv expression vector into Corynebacterium glutamicum, and culturing the respective recombinant strains.
FIG. 3 shows the results of comparing the expression efficiency of Rluc8 by transforming each of the Rluc8 secretion expression vectors to which 14 signal sequences have been applied into Corynebacterium glutamicum, and culturing the respective recombinant strains.
FIG. 4 shows the results of comparing secretion expression efficiency of asparaginase by transforming each of the secretory expression vectors of asparaginase to which 14 signal sequences were applied into Corynebacterium glutamicum, and culturing the respective recombinant strains.
Figure 5 shows the expression and secretion of asparaginase in a recombinant strain of Corynebacterium glutamicum transformed with the recombinant vector for expression of asparaginase (pXMJ19 / ssNCgl2629-ansB) to which the NCgl2629 signal sequence was applied to induce the growth of the recombinant strain And the accumulation of asparaginase in the medium.
FIG. 6 shows the expression of asparaginase secreted by culturing a recombinant strain of Corynebacterium glutamicum transformed with pXMJ19 / ssNCgl2629-ansB in a fermenter, showing the growth of the recombinant strain and the accumulation of asparaginase in the medium and productivity .
FIG. 7 shows the results of SDS-PAGE analysis of fractions obtained by separating and purifying asparaginase secreted from the medium through FPLC using an anion exchange resin.
FIG. 8 shows the productivity of a recombinant strain having a signal sequence to which the lamp theory is applied.
FIG. 9 shows the result of analysis of 2-DE (2-Dimensional Electrophoresis) analysis of asparaginase produced and purified from recombinant Corynebacterium glutamicum.

Hereinafter, the present invention will be described in detail. The terms used in the present specification should be construed as generally understood by a person having ordinary skill in the art unless otherwise defined. It is to be understood that the drawings and embodiments are not intended to limit the scope of the present invention, which is not intended to limit the scope of the present invention. It is not.

The present invention is an invention for Corynebacterium (Corynebacterium) the foreign protein is asparaginase recombinant vector, transformed by Corynebacterium and asparaginase production methods using them to produce the (L-asparaginase) in.

Currently, recombinant E. coli is mainly used to produce asparaginase. However, when the protein is isolated and purified from recombinant E. coli, it is difficult to completely remove the contaminants that may act as an endotoxin in animals. In addition, changes in structure or activity of the protein may occur during post-translational modification (PTM) of the host and side reactions that may occur during the separation and purification process. Indeed, it has been reported that variants of asparaginase contained in commercially available formulations are mixed. It is a very important task to prevent the transformation of the structure or activity of the protein produced by the strain because serious side effects may occur when such a modified protein is used for medicinal use and the like.

The present invention can fundamentally solve the problems occurring in the production of asparaginases using existing strains using Corynebacterium. Specifically, Corynebacterium used for the secretion and production of asparaginase in the present invention has a low extracellular proteolytic activity and is advantageous for excretion of proteins externally because of its simple membrane structure. Since Corynebacterium is a generically recognized as safe (GRAS) strain and is harmless to the human body, it can solve the problem caused by endotoxin, which is a problem when using Gram-negative bacteria. Therefore, And so on. Despite these useful features, studies to secrete and produce exogenous proteins using Corynebacterium have been performed only on very few proteins and are not commonly used for protein production. For example, Corynebacterium has various secretory signal sequences, but only secretory sequence signals of PS, which is one of secretory proteins of Corynebacterium, are mainly used.

In order to use a strain of the genus Corynebacterium as an asparaginase producing strain, the present invention provides a recombinant vector comprising asparaginase gene bound to any one of several signal sequences derived from Corynebacterium to provide. The signal sequence of Corynebacterium is a sequence for secretion of the protein originally expressed in Corynebacterium. After the protein is expressed, it is secreted out of the cell according to various mechanisms by the signal sequence (signal peptide). However, asparaginase is a heterologous protein that is not originally expressed in Corynebacterium, and is not secreted extracellularly by the binding of the signal sequence derived from Corynebacterium. In addition, even when a suitable promoter, vector, or the like is used to transform Corynebacterium, there is no guarantee that the asparaginase gene to which the signal sequence binds is always expressed as a normal asparaginase protein. That is, it is important to identify the Corynebacterium signal sequence capable of normal expression and secretion of the asparaginase gene bound to the Corynebacterium signal sequence, and provides a signal sequence consistent with the present invention.

According to one embodiment of the present invention, the degree and degree of secretion of Corynebacterium may vary depending on the type of signal sequence binding to the N-terminal of asparaginase. Asparaginase expression is high but there is also a signal sequence that is not secreted. In other proteins besides asparaginase, protein expression and secretion occur well, but asparaginase also has a signal sequence that does not show the same effect.

In the present invention, Corynebacterium can be used without limitation as long as it is a strain belonging to genus Corynebacterium. For example, Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium ammoniagenes to Ness (Corynebacterium ammoniagenes), Corynebacterium Kasei (Corynebacterium casei ) and Corynebacterium efficiens may be used as the strain of the genus Corynebacterium.

In the present invention, the asparaginase is Escherichia but are not limited to, proteins derived from E. coli or Erwinia carotovora . In addition, new asparaginases derived from plants, animals and microorganisms with reduced glutaminase activity can be produced. In addition, production and recovery of useful proteins such as protease, aminopeptidase, carboxypeptidase, collagenase and chitinase can be also applied by applying the technique of the present invention.

In the present invention, a general Corynebacterium expression vector may be used as a vector used for expression of asparaginase. Specifically, the expression vector may include a promoter, a multiple cloning site, a gene expression control sequence, a replication origin sequence for vector maintenance, and a typical expression vector suitably containing an antibiotic marker gene. In order to secrete and express the target protein in the expression vector, a suitable signal sequence is included at the N-terminus, and the recombinant strain can be formed through transformation of Corynebacterium.

The vector that can be used in the present invention is not particularly limited as long as it can function in Corynebacterium. It can be inserted into and maintained in the chromosome as well as the plasmid form. Examples of the expression vector include pEKEx1, pXMJ19, pCES208, pCRA1, pCRA429, pDXW-8, pBKGEXm2, pZ8-1, pVWEx1, pSK360, pEC901, pEC-XK99E, pTRCmob, pAPE12, pXK99E, pHM1519, pAM330, Can be selected and used. In addition, an artificial transposon may be used, and when a transposon is used, a desired gene may be introduced into the chromosome.

The promoter that can be used in the present invention is not particularly limited, and a heterologous host-derived promoter such as E. coli-derived tac may be used. For example, the promoter of the gene of PS1, PS2, SlpA of the cell surface protein, the amino acid biosynthesis system of various amino acids, the glutamate dehydrogenase gene of glutamic acid biosynthesis system, the glutamine synthase gene of glutamine synthesis system, A homoserine dehydrogenase gene of a threonine biosynthesis system, an acetohydroxy acid synthase gene of an isoleucine and a valine biosynthetic system, a 2-isopropylmalate synthase gene of a leucine biosynthesis system, a glutamate kinase of a proline and arginine biosynthesis system, (DAHP) synthase gene of an aromatic amino acid biosynthesis system such as a phospholiposyl-ATP pyrophosphorylase gene of a histidine biosynthetic system, tryptophan, tyrosine and phenylalanine, a nucleic acid such as inosine acid and guanylic acid In the biosynthetic system, phosphoribosyl pyrophosphate ( PRPP) amidotransferase gene, an inosine dehydrogenase gene, and a promoter of a guanylate synthase gene. In addition, a promoter that regulates essential genes of Corynebacterium can also be used, and typically there is a promoter of a housekeeping gene. Corynebacterium has a total of seven sigma factors (SigA, SigB, SigC, SigE, SigH, and SigM), mainly SigA, and most housekeeping genes are transcribed by SigA and RNAP complexes. Therefore, a promoter recognized by SigA can be used.

In the present invention, a method for introducing an expression vector into a host is not particularly limited, and known techniques can be used.

In the present invention, the signal sequence binding to the N-terminus of asparaginase can be a signal sequence derived from Corynebacterium as an expression host. The signal sequence of the cell surface protein or secretory protein in the signal sequence of Corynebacterium is preferably, but not limited thereto. In addition, signal sequences of surface proteins including estrase, porine B, oxidase, and phosphodiesterase may be used in the present invention.

According to one embodiment of the present invention, signal sequences of PS1 and PS2 which are typical secretory proteins in Corynebacterium can be used as signal sequences.

According to another embodiment of the present invention, when the signal sequence is the same but the reporter protein is not expressed or secreted when it is bound to a gene encoding a reporter protein, expression and secretion of asparaginase may well occur. In addition, expression and secretion may occur well regardless of the type of protein to which the signal sequence binds.

According to the present invention, the signal sequence bound to the target protein is cleaved by the signal peptidase when the target protein is secreted outside the cell, and is mostly removed. However, in some cases, a portion of the signal sequence may remain at the N-terminus, which may result in a functional folding of the protein. To solve this problem, a technique of combining a ramp-tag into a signal sequence can be used.

According to one embodiment of the present invention, it is possible to transform asparaginase having an optimal codon at the time of protein expression through the lamp tag technology, thereby recovering an excellent form of asparaginase that does not undergo transformation during the expression, secretion and purification of asparaginase .

In order to separate, purify, and recover the asparaginase accumulated in the medium after culturing the recombinant Corynebacterium strain for secretion and expression of the asparaginase of the present invention, various known methods can be used. For example, the microbial cells can be removed by centrifugation or the like and then purified by salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, medium pressure liquid chromatography, reversed phase chromatography, And the like, or a combination thereof.

According to one embodiment of the present invention, asparaginase can be produced at a high yield by a simple method, and the recovery rate is also excellent. Unlike the purification and recovery of proteins expressed and secreted from Escherichia coli recombinant strains, a process for removing contaminants such as host cells and modified proteins may not be performed.

The production of asparaginase using the Corynebacterium recombinant strain of the present invention is suitable for large-scale production for commercial purposes as well as on a laboratory scale.

According to one embodiment of the present invention, the productivity and the recovery rate of the asparaginase hardly decrease even in a large scale production.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not construed as being limited thereto.

In all of the examples of the present invention, Escherichia coli for gene cloning and Corynebacterium for asparaginase production were cultured and transformed in the following manner.

Escherichia coli: E. coli) is XL1-Blue (prepare JM110) by gene cloning host, and Corynebacterium is Corynebacterium glutamicum (Corynebacterium glutamicum: C. glutamicum) ATCC13032 (American Type Culture Collection) for asparagus It was prepared for the production of laginase. Escherichia coli and Corynebacterium glutamicum strains were cultured at 37 ° C and 32 ° C using LB (Luria-Bertani, difco) medium. When necessary, appropriate amounts of antibiotics (kanamycin 20 μg / ml, chloramphenicol 10 mu g / ml, Ampicillin 50 mu g / ml).

To the competent cells (competent cell) Preparation of E. coli XL1-Blue (JM110), first, a single colony and incubated the cells on LB plate medium was inoculated in a liquid pre-culture medium and the addition of MgCl ₂ 20㎖ SOB medium Were cultured at 22 < [deg.] ≫ C. After incubation, OD _600nm value was recovered at 0.5, treated with Inoue transformation buffer, and stored at -80 ° C. For transformation, each recombinant DNA was mixed with competent cells according to a conventional heat shock method, and then allowed to stand on ice for 30 minutes, followed by thermal shock at 42 ° C for 90 seconds and left on ice for 2 minutes . After that, the cells were cultured at 37 ° C., streaked on LB solid medium containing appropriate antibiotics, and grown for 15-20 hours. Colony cracking and colony PCR were performed to obtain a clone into which the desired gene was inserted.

Competent cells of Corynebacterium glutamicum ( C. glutamicum ) were prepared as follows. First, the cells were cultured in an LB plate culture medium. Then, a single colony was inoculated on an LB liquid medium containing 2% glucose and pre-cultured. The culture solution was added with 4 / / ml of isonicotinic acid hydrazide (isoniazid) / Ml and Tween 80 (0.1% Tween 80), incubated at 25 ° C, and recovered at an OD _600nm value of 0.6. The cells were recovered by 4 times with 10% glycerol After electrophoresis, cells were washed with 100 μl aliquots and stored at -80 ° C.

In order to increase the yield of the coryneform bacteria glutamicum transformation method, recombinant vector without methylation pattern was recovered after transformation into JM110. In order to transform the coryneform bacteria glutamicum by electroporation, 200-500 ng / mu l of a recombinant plasmid was added to 100 mu l of cells, and the transformants were washed with 0.2 cm cuvette, 25 uF, 600 ohm, 2.5 kV, (Bio-Rad, USA), and then immediately subjected to thermal shock for 6 minutes at 46 ° C. Then, 1 ml of SOC medium was added, and cultured at 32 ° C for 1.5 hours. The recombinant strains were selected by streaking on LB solid medium containing appropriate antibiotics.

In order to compare the degree of secretion of protein by secretory signal sequences in Corynebacterium strains, the expression and secretion levels of reporter proteins GFPuv and Rluc8 were compared.

As a sequence signal of Corynebacterium strain for confirming expression and secretion of protein, Corynebacterium ( Corynebacterium < RTI ID = 0.0 > ammoniagenes derived CspA, Corynebacterium < RTI ID = 0.0 > glutamicum ATCC14067) was used as the origin PS2, NCgl0336, NCgl0932, NCgl1930, NCgl2101, NCgl2779, NCgl2865, NCgl2185, NCgl1331, NCgl0801, NCgl2629. In addition to the heterologous sequence signal wave Enigma Bacillus (Paenibacillus sp.) Derived from Psp, L. monocytogenes cytokines to Ness (Listeria monocytogenes) was used as the origin inlA. The gene sequence information of the used signal sequence is shown in [Table 1].

gene Explanation Signal sequence (/, cleavage site) type SEQ ID NO: cspA Cell surface proteinA MKRMKSLAAALTVAGAMLAAPV / ATA
Sec SEQ ID NO: 1 cspB PS2 MFNNRIRTAALAGAIAISTAASGVAIP / AFA Sec SEQ ID NO: 2 psp Endo-celluase MGLKMKKRSGKKAWMLLVMSLLIAAVPIT / ASA Sec SEQ ID NO: 3 inlaid InternalinA MRKKRYVWLKSILVAILVFGSGVWINTSNGTN / AQA Sec SEQ ID NO: 4 NCgl0336 Esterase MKLLRRIAAPAIALGIAMSTIVTPST / AGA Sec SEQ ID NO: 5 NCgl0932 PorB MKKLRFATIAAATVALTASLTPS / ASA Sec SEQ ID NO: 6 NCgl1930 Hypothetical protein MRKFRNTAIALVSAAAITLGG / VTA Sec SEQ ID NO: 7 NCgl2101 Similar to esterase MRKGISRVLSVAVASSIGFGTVLTGTGI / AAA Sec SEQ ID NO: 8 NCgl2779 Esterase MSVFTRAGEASRKLVALVVALATAAALMVVGQGT / AQA Sec SEQ ID NO: 9 NCgl2865 Multicopper oxidase MTSSFSRRQFLLGGLVLAGTGAVAACTSDPGP / AAS Taste SEQ ID NO: 10 NCgl2185 Phosphodiesterase MRSLMPQLSRRQFLQTTAVTAGLATFAGTP / ARA Taste SEQ ID NO: 11 NCgl1331 ABC transporter periplasm component MAQISRRHFLAAATVAGAGATLA / ACA Taste SEQ ID NO: 12 NCgl0801 Hypothetical protein MQINRRGFLKATTGLATIGAASMFMPK / ANA Taste SEQ ID NO: 13 NCgl2629 Hypothetical membrane protein MVNTLNSKTVNVPRFARGVVAAATALFFGALVSLAPS / ALA Taste SEQ ID NO: 14

For the production of the recombinant vector, pXMJ19 was used as a Corynebacterium expression vector. GFPuv and Rluc8 genes were amplified by PCR using the primers shown in Table 2 below. Each reporter gene was then cloned into the pXMJ19 vector using HindIII and KpnI restriction sites of the vector. As a result, pXMJ19 / GFPuv and pXMJ19 / Rluc8 recombinant vectors were prepared. The HindIII restriction site of the recombinant vector was then replaced with EcoRV to insert the signal sequence. The circular PCR method was used as a substitution method, and the primers of the sequence signals used in the preparation of the recombinant vector are shown in Table 3 below.

primer The sequence (5'-3 ') Restriction enzyme site SEQ ID NO: gfpuv F CCAAAGCTTATGGCATGCCTGCAGGTCGACTC HindIII SEQ ID NO: 15 gfpuv R CCAGAATTCTTATTTGTACAGCTCATCC EcoRI SEQ ID NO: 16 Rluc8_19 F AAAAAGCTTATGGCTTCCAAGGTGTACG HindIII SEQ ID NO: 17 Rluc8_19 R AAAGAATTCTCAATGATGATGATGATGATGGTCG EcoRI SEQ ID NO: 18 Rluc8 F
(ecoRV) ATAGATATCATGGCTTCCAAGGTGTACGACC EcoRV SEQ ID NO: 19 gfpuv F
(ecoRV) ATAGATATCGCATGCCTGCAGGTCGACTC EcoRV SEQ ID NO: 20 plaC R
(ecoRV) ATAGATATCAATTAATTCTGTTTCCTGTGTGAAATTGTTATCC EcoRV SEQ ID NO: 21

primer The sequence (5'-3 ') SEQ ID NO: cspA F ATGAAACGCATGAAATCGCTGG SEQ ID NO: 22 cspA R TTTTTCTGCTGCCGTTGCC SEQ ID NO: 23 cspB F ATGTTTAACAACCGTATCCGCACTG SEQ ID NO: 24 cspB R GGTTTCCTGAGCGAATGCTGG SEQ ID NO: 25 psp F ATGGGTTTGAAGATGAAGAAAAGATCAG SEQ ID NO: 26 psp R ATCCGATGCTGCGCTGG SEQ ID NO: 27 inlaid F ATGAGAAAAAAACGATATGTATGGTTGAAAAG SEQ ID NO: 28 R AATTGTAGCTGCCTGAGCATTTG SEQ ID NO: 29 0336 F ATGAAGCTTCTTCGCCGCATC SEQ ID NO: 30 0336 R TACTTCGGCAGCGCCTGC SEQ ID NO: 31 0932 F ATGAAGAAACTACGTTTCGCCACC SEQ ID NO: 32 0932 R GAAATCCTGTGCGGAAGCTGAG SEQ ID NO: 33 1930 F ATGCGTAAATTCCGCAACACTG SEQ ID NO: 34 1930 R AGTTTCGTCTTCCTGAGCGGTAG SEQ ID NO: 35 2101 F ATGCGTAAAGGAATTTCCCGCG SEQ ID NO: 36 2101 R AGAGTCTTGAGCTGCTGCGATG SEQ ID NO: 37 2779 F ATGTCCGTATTTACACGAGCTGG SEQ ID NO: 38 2779 R ACGGTTTGCAGCTTGTGC SEQ ID NO: 39 2865 F ATGACAAGTAGTTTTTCCCGGCG SEQ ID NO: 40 2865 R ACCTGGTGCCGAGGCAG SEQ ID NO: 41 0801 F ATGCAAATAAACCGCCGAGGC SEQ ID NO: 42 0801 R TGCTCCAAGGGCGTTGGC SEQ ID NO: 43 2185 F ATGCCACAGTTAAGCAGACGC SEQ ID NO: 44 2185 R GCGTTCTTCAGCGCGTGC SEQ ID NO: 45 2629 F ATGGTGAACACGTTGAACTCTAAAACC SEQ ID NO: 46 2629 R TGGTTCCTGCGCCAACG SEQ ID NO: 47 1331 F ATGGCTCAGATTTCTCGTCGTC SEQ ID NO: 48 1331 R ACCGGTACCCGCACATG SEQ ID NO: 49

Each of the 14 signal sequences was amplified by PCR from chromosomal DNA and the amplified signal sequence genes were blunt-ended joined to the EcoRV restriction site of the pXMJ19 / GFPuv and pXMJ19 / Rluc8 recombination vectors. Lt; / RTI > As a result, the signal sequences of ssCspA-, ssCspB-, ssPsp-, ssinlA-, ssNCgl0336-, ssNCgl0932-, ssNCgl1930-, ssNCgl2101-, ssNCgl2779-, ssNCgl2865-, ssNCgl2185-, ssNCgl1331-, ssNCgl0801- and ssNCgl2629- A recombinant vector expressing GFPuv and Rluc8 was constructed. The production scheme of the recombinant vector to which the signal sequence is applied is as follows (FIG. 1). The recombinant vectors produced by the above method are shown in Table 4 below.

Plasmid Explanation size pXMJ19 / gpfuv GFPuv expression, Ptac, CmR 7323 bp pXMJ19
/ ssCspA-gfpuv GFPuv secretion, Ptac, CmR 7407 bp pXMJ19
/ ssCspB-gfpuv GFPuv secretion, Ptac, CmR 7422 bp pXMJ19
/ ssPsp-gfpuv GFPuv secretion, Ptac, CmR 7428 bp pXMJ19
/ ssInlA-gfpuv GFPuv secretion, Ptac, CmR 7437 bp pXMJ19
/ ss0336-gfpuv GFPuv secretion, Ptac, CmR 7419 bp pXMJ19
/ ss0932-gfpuv GFPuv secretion, Ptac, CmR 7410 bp pXMJ19
/ ss1930-gfpuv GFPuv secretion, Ptac, CmR 7419 bp pXMJ19
/ ss2101-gfpuv GFPuv secretion, Ptac, CmR 7425 bp pXMJ19
/ ss2779-gfpuv GFPuv secretion, Ptac, CmR 7443 bp pXMJ19
/ ss2865-gfpuv GFPuv secretion, Ptac, CmR 7437 bp pXMJ19
/ ss2185-gfpuv GFPuv secretion, Ptac, CmR 7419 bp pXMJ19
/ ss1331-gfpuv GFPuv secretion, Ptac, CmR 7410 bp pXMJ19
/ ss0801-gfpuv GFPuv secretion, Ptac, CmR 7422 bp pXMJ19
/ ss2629-gfpuv GFPuv secretion, Ptac, CmR 7452 bp pXMJ19 / rluc8 Rluc8 expression, Ptac, CmR 7516 bp pXMJ19
ssCspA-rluc8 Rluc8 secretion, Ptac, CmR 7600 bp pXMJ19
ssCspB-rluc8 Rluc8 secretion, Ptac, CmR 7615 bp pXMJ19
ssPsp-rluc8 Rluc8 secretion, Ptac, CmR 7621 bp pXMJ19
ssInla-rluc8 Rluc8 secretion, Ptac, CmR 7630 bp pXMJ19
ss0336-rluc8 Rluc8 secretion, Ptac, CmR 7612 bp pXMJ19
ss0932-rluc8 Rluc8 secretion, Ptac, CmR 7603 bp pXMJ19
ss1930-rluc8 Rluc8 secretion, Ptac, CmR 7612 bp pXMJ19
ss2101-rluc8 Rluc8 secretion, Ptac, CmR 7618 bp pXMJ19
ss2779-rluc8 Rluc8 secretion, Ptac, CmR 7636 bp pXMJ19
ss2865-rluc8 Rluc8 secretion, Ptac, CmR 7630 bp pXMJ19
ss2185-rluc8 Rluc8 secretion, Ptac, CmR 7612 bp pXMJ19
ss1331-rluc8 Rluc8 secretion, Ptac, CmR 7603 bp pXMJ19
ss0801-rluc8 Rluc8 secretion, Ptac, CmR 7615 bp pXMJ19
ss2629-rluc8 Rluc8 secretion, Ptac, CmR 7645 bp

The prepared recombinant vector was transformed into Corynebacterium glutamicum strain as described in Example 1 to prepare respective recombinant strains, and the expression and secretion degree of the reporter protein were measured according to the signal sequence. For measurement of secretory expression, Corynebacterium glutamicum recombinant strains were cultured in LB medium, respectively. When the absorbance (OD _600nm ) was about 0.75-1.0, 0.5 mM IPTG was added to induce the expression of the recombinant protein. After further incubation for 3 hours, cells and medium were recovered and the expression and secretion level of the reporter protein were confirmed by SDS-PAGE and reporter activity measurement.

As shown in the results of SDS-PAGE and GFPuv fluorescence analysis (infinite M200, TECAN), when the ssNCgl0932-, ssNCgl2865-, ssNCgl1331-, and ssNCgl0801- signal sequences were applied, relatively good expression and secretion , And it was confirmed that secretion was relatively well secreted through the Tat-dependent pathway rather than the Sec-dependent pathway (Fig. 2). In particular, when the ssNCgl0801- signal sequence was applied, the highest fluorescence value was confirmed in the medium, and the SDS-PAGE analysis confirmed a clear protein band in the protein-secreted medium. On the other hand, when ssCspA, ssPsp-, and ssNCgl1331- were applied, overexpression was observed in the cells, but the ratio of the protein secreted to the medium was low. These results suggest that secretory signal sequences such as ssCspA, ssPsp-, and ssNCgl1331- may be helpful for the expression of GFPuv, but secretion function may be lost or inhibited by binding with foreign proteins.

In case of Rluc8, secretion efficiency was confirmed by SDS-PAGE and luciferase enzyme activity measurement as in GFPuv. The luciferase enzyme activity of Rluc8 was confirmed by measuring luminescence at 562 nm after reacting in 96 well microplates using coelenterazine as a substrate. As a result, it was confirmed that the ssNCgl2185- and ssNCgl0801- signal sequences were relatively well expressed and secreted, and it was confirmed that secretion was relatively well secreted through the Tat-dependent pathway rather than the Sec-dependent pathway as in GFP (Fig. 3) . On the other hand, ssPsp- and ssNCgl2629- were overexpressed in the cytoplasm but not secreted. These results also confirm that secretory signal sequences such as ssPsp- and ssNCgl2629- can help to express Rluc8, but secretion function can be lost or inhibited by binding with foreign proteins.

The expression and secretion level of Escherichia coli-derived asparaginase II (L-asparaginase II) was determined in the recombinant Corynebacterium strain according to the signal sequence.

The asparaginase gene was amplified by PCR from E. coli chromosomal DNA. As shown in Example 2, the pXMJ19 vector was cloned using HindIII and KpnI restriction enzyme sites and ligated to EcoRV restriction enzyme site , And the signal sequence amplified by PCR was inserted into the EcoRV site to prepare a recombinant vector to which each signal sequence was applied (see FIG. 1). The primers used in the preparation are shown in Table 5, and the prepared recombinant vectors are shown in Table 6 below.

primer The sequence (5'-3 ') Restriction enzyme site SEQ ID NO: Asp_19 F CCTAAGCTTATGGAGTTTTTCAAAAAGACGGC HindIII SEQ ID NO: 50 Asp_19 R ACTGAATTCTTAGTACTGATTGAAGATCTGCTGG EcoRI SEQ ID NO: 51 asp F (ecoRV) ATAGATATCATGGAGTTTTTCAAAAAGACGGCACTTG EcoRV SEQ ID NO: 52 plac R (ecoRV) ATAGATATC AATTAATTCTGTTTCCTGTGTGAAATTGTTATCC EcoRV SEQ ID NO: 53

Plasmid Explanation Size (bp) pXMJ19 / ansB Asparaginase expression, Ptac, CmR 7537 bp pXMJ19
/ ssCspA-ansB Asparaginase secretion, Ptac, CmR 7621 bp pXMJ19
/ ssCspB-ansB Asparaginase secretion, Ptac, CmR 7636 bp pXMJ19
/ ssPsp-ansB Asparaginase secretion, Ptac, CmR 7642 bp pXMJ19
/ ssInla-ansB Asparaginase secretion, Ptac, CmR 7651 bp pXMJ19
/ ss0336-ansB Asparaginase secretion, Ptac, CmR 7633 bp pXMJ19
/ ss0932-ansB Asparaginase secretion, Ptac, CmR 7624 bp pXMJ19
/ ss1930-ansB Asparaginase secretion, Ptac, CmR 7633 bp pXMJ19
/ ss2101-ansB Asparaginase secretion, Ptac, CmR 7639 bp pXMJ19
/ ss2779-ansB Asparaginase secretion, Ptac, CmR 7657 bp pXMJ19
/ ss2865-ansB Asparaginase secretion, Ptac, CmR 7651 bp pXMJ19
/ ss2185-ansB Asparaginase secretion, Ptac, CmR 7633 bp pXMJ19
/ ss1331-ansB Asparaginase secretion, Ptac, CmR 7624 bp pXMJ19
/ ss0801-ansB Asparaginase secretion, Ptac, CmR 7636 bp pXMJ19
/ ss2629-ansB Asparaginase secretion, Ptac, CmR 7666 bp

Then, as described in Example 2, each recombinant strain of Corynebacterium glutamicum was cultured, and the secretion efficiency of the asparaginase expressed and secreted in the medium was measured by SDS-PAGE and enzyme activity measurement. The enzymatic activity of asparaginase was determined by a general asparaginase enzyme activity assay using Nessler's reagent. As a result, it was confirmed that ssCspA-, ssCspB-, ssPsp-, ssNCgl0336-, ssNCgl2101-, and ssNCgl2629- were relatively well expressed and secreted, and the asparaginase activity of the medium was high (FIG. 4). In addition, it was confirmed that the expression and secretion were good in the overall Sec- and Tat-dependent signal sequence, unlike the results of GFPuv and Rluc8. SDS-PAGE of the medium showed the highest efficiency of asparaginase secreted in the medium for the same time when the ssNCgl2629- signal sequence was applied.

In Example 2 and Example 3, secretion efficiencies of different kinds of protein (GFPuv, Rluc8, asparaginase) and signal sequences were compared in the expression and secretion of heterologous proteins. From the results of the comparison, it was confirmed that the expression and secretion efficiency could be varied depending on the type of the signal sequence and the type of the target protein. In the case of the reporter protein, it has been confirmed that the secretion expression efficiency is generally high due to the Tat-dependent signal sequence. This is because, since the proteins originate from the functional folding of the proteins in the cytoplasm, It is presumed that the signal sequence was more suitable for secretion expression. Conversely, in the case of asparaginase, it is found that Sec- and Tat-dependent signal sequences are relatively well applied to the surface proteins of origin. Of the 14 signal sequences, the ssNCgl0801- signal sequence was found to be secreted in all cases (GFPuv, Rluc8, asparaginase). Therefore, it was found to be most advantageous to use the ssNCgl0801- sequence for the secretory expression of the heterologous protein using Corynebacterium. From the above results, it was confirmed that the recombinant strain of Corynebacterium glutamicum could be used for the overproduction of asparaginase through secretion and expression.

Asparaginase expression and secretion production using corynebacterium was increased by scaling up in LB medium to produce asparaginase.

A recombinant strain of Corynebacterium glutamicum transformed with pXMJ19 / ssNCgl2629-asnB was cultured in a 5 L Erlenmeyer flask with 1 L of LB medium. The culture conditions were incubated at 32 ° C and 200 rpm for 24 hours. Absorbance was measured. When the absorbance (OD _600nm ) was about 0.75-1.0, 0.5 mM IPTG was added to induce the expression of the recombinant protein. Subsequently, the medium was recovered at intervals of 2 hours to analyze the asparaginase secreted by the medium, and SDS-PAGE analysis and asparaginase enzyme activity were measured. As a result, it was confirmed that the amount of asparaginase accumulated in the medium after the induction of protein expression and the activity of asparaginase increased proportionally with the accumulation amount. As can be seen from the results of SDS-PAGE analysis, there were not many kinds of secretory proteins secreted by Corynebacterium glutamicum in the medium, and it was confirmed that about 80% or more of total secreted proteins were asparaginase proteins (Fig. 5). Therefore, it was confirmed that the target protein can be effectively recovered by minimizing the contaminating protein in the separation and purification process.

An experiment was conducted to confirm whether recombinant strains could be overproduced by culturing the recombinant strains under optimal growth conditions using a fermenter and increasing the cell mass of the recombinant strains. The culture conditions of pXMJ19 / ssNCgl2629-asnB Corynebacterium glutamicum recombinant strains were as follows. 10 g (NH ₄ ) ₂ SO ₄ , 3 g KH ₂ PO ₄ , 3 g Yeast extract, 5 g of dextrose in a 5 L fermenter (Jar Bioreactor; BioCNS) containing dextrose as a carbon source MgSO ₄揃 7H ₂ O, 10 mg FeSO ₄揃 7H ₂ O, 10 mg MnCl ₂揃 4H ₂ O, 10 mg ZnSO ₄揃 7H ₂ O, 1 mg CuCl ₂揃 4H ₂ O, 0.1 mg Biotin, 1 mg Thiamin per liter with Chloramphenicol 10 mg / L) was inoculated with 1% (v / v) of a recombinant strain pre-cultured. The culture was carried out at 32 ° C, pH 7.0, 200-800 rpm, and protein expression was induced under the same conditions as in the flask experiment, followed by further incubation for about 20 hours. During culture, the pH of the medium was maintained using a 50% Ammonia solution, the air flow was maintained at 1.0 vvm and bubbles were minimized during incubation with antiform B emulsion . Subsequently, the medium was recovered at intervals of 2 hours to analyze the asparaginase secreted by the medium, and SDS-PAGE analysis and asparaginase enzyme activity were measured. As a result, no remarkable stress was observed in the cells when cultured under the above conditions, and it was confirmed that the cell absorbance (OD _600nm ) was 110.4 at 14 hours after culturing and that the cells grew well to very high concentration. SDS-PAGE analysis revealed that aspartaginase protein bands were evident in the medium from about 4 hours after induction of asparaginase expression using IPTG. As the cell concentration and incubation time increased, the amount of asparaginase Of the total number of patients. In addition, about 50% of the total secreted protein was found to be asparaginase, and when the cells were recovered and the accumulation of asparaginase was confirmed in the cells, it was found that asparaginase was not accumulated in the cells, (Fig. 6).

As a result, the expression and secretion efficiency of asparaginase were maintained and the secretion of asparaginase accumulated in the medium was normal.

After the cultivation of the recombinant Corynebacterium, the asparaginase secreted by the medium was separated and purified. In purification, contamination of impurities due to cell crushing is not a problem, so pure asparaginase of high purity can be separated by a relatively simple purification process.

First, the cells were removed by centrifugation (10,000 x g, 20 minutes) for separation and purification of asparaginase in the medium. Then, the medium was filtered using a 0.45 mu m filter to remove insoluble matter. A typical anion-exchange culumn was used for protein separation in 30 ml of the prepared culture medium. To this end, the filtered medium was suspended in 120 ml of a binding buffer (20 mM Tris-HCl, pH 6.5) and further centrifuged to remove insoluble matters. Then, the anion exchange resin (HiTrap Q HP, GE Healthcare, 5 ml) was washed with 50 ml of a binding buffer to reach equilibrium, and the prepared medium suspension was flowed at a flow rate of 2 ml / min to induce protein adhesion After that, 100 ml of a binding buffer was further flowed at a flow rate of 4 ml / min to remove proteins that were not bound to the column and were not bound specifically to the column. Then, the eluted buffer (elution buffer, 20 mM Tris-HCl, pH 6.5, 1.0 M NaCl) was flowed at a flow rate of 1 ml / min to recover proteins adsorbed on the anion exchange resin. At this time, all of the solutions that flowed in each process were recovered to confirm the degree of separation and purification from other contaminating proteins. After the aspartase recovered fractions were identified by SDS-PAGE analysis (FIG. 7), a buffer having a molecular weight cut-off (MWCO) of 10 KDa was used to remove the high concentration of sodium chloride used in the elution process. Exchange. When the asparaginase is purified through the above procedure, the yield of the purification is as shown in Table 7 below. However, since the culture medium has the property of being secreted by the medium, the culture and purification can be continuously performed, and the purification efficiency can be further improved.

Asparaginease productivity Recovery rate NCgl2629-ansB 114 mg / L > 30%

As a method of promoting the expression rate of asparaginase and functional folding of proteins, the gene sequence coding for the signal sequence was incorporated into the lamp technology to improve the productivity of secreted asparaginase.

The codon usage of Corynebacterium glutamicum ATCC13032 strain was analyzed for the application of lamp tag technology to the signal sequence and rare codons were collected. The rare codons referred to here refer to the codons that are collected in the process described below, in addition to the concept of commonly known rare codons. The rate of translation is most influenced by how rapidly the aa-tRNA, in which the amino acid is bound, is released when the codon is decoded. Therefore, the rate of translation of aa-tRNA at a codon in which the amount of the aa-tRNA is sufficiently present in the cell is fast, while the lack of aa-tRNA decreases translation efficiency. Therefore, the gene copy of tRNA can be used as the first measure of the concentration of the corresponding tRNA in the cell. As is known, a typical codon optimization method reflects the codon frequency because it is known that the codon frequency is proportional to the number of tRNA copies. In this experiment, codon frequencies (100% standard) and tRNA gene copies corresponding to each amino acid (AA) were used in Corynebacterium glutamicum (http://gtrnadb.ucsc.edu/), and rare codons were collected (KR 20130027549). ≪ / RTI > Briefly, the codon pair frequencies in each amino acid were less than 1.0%, and up to three codons for one amino acid were collected (Table 8). The method of considering each codon frequency and the method of considering the gene copy number of isoacceptor tRNA were mixed.

Amino acid (AA) Codon frequency tRNA His CAC 0.77 Ile AUA 0.18 Tyr UAU 0.70 Cys UGU 0.20 UGC 0.41 Gly GGG 0.67 One Pro CCC 0.97 One Thr ACG 0.88 One ACA 0.76 One Val GUA 0.81 One Ser AGU 0.49 UCG 0.76 One Arg CGG 0.49 One CGA 0.66 AGG 0.32 One AGA 0.22 One Leu CUA 0.58 One UUA 0.51

Indication of gene Secretory signal sequence SEQ ID NO: R1-ss2629 GTAAATACACTCAATAGTAAAACGGTCAATGTACCTCGGTTTGCACGGGGCGTAGTAGCCGCTGCGACAGCGTTATTTTTCGGGGCCCTAGTTTCGCTTGCCCCCTCGGCGCTAGCG SEQ ID NO: 54 R2-ss2629 Gt; SEQ ID NO: 55 R3-ss2629 GTAAATACGTTAAACAGTAAGACGGTGAATGTACCGAGATTCGCAAGAGGTGTGGTAGCGGCAGCCACGGCGCTATTCTTCGGTGCTCTTGTAAGTCTTGCACCCTCGGCCCTAGCA SEQ ID NO: 56 R4-ss2629 Gt; SEQ ID NO: 57

The recombinant asparaginase having the secretion sequence prepared according to the method described above was expressed in Corynebacterium and the solubility thereof was evaluated. The pXMJ19 recombinant vector in which the ramp-tagged secretion sequence ([Table 9]) was cloned was transformed into Corynebacterium glutamicum as described in Example 1. Then, as described in Example 1, the recombinant strains of Corynebacterium glutamicum were separately cultured, and the secretion efficiency of asparaginase expressed by the medium was measured by SDS-PAGE and enzyme activity. The enzymatic activity of asparaginase was determined by a general asparaginase enzyme activity assay using Nessler's reagent. As a result of the experiment, it was confirmed that the recombinant strains having the ss2629-secretion sequence expressed more secretion than the R2-ss2629- and R3-ss2629-, respectively. When R1-ss2629- and R4-ss2629- were applied, There was no. It was confirmed that asparaginase activity of the medium was high when R2-ssNCgl2629- and R3-ssNCgl2629- were applied (Fig. 8). Therefore, it was confirmed that the ss2629-secretory sequence redesigned by the lamp tag increases the expression rate

Also, in order to confirm production of asparaginase using a fermenter, a recombinant strain was cultured in a 2 L defined medium under the same culture conditions as in Example 4. Then, the medium was recovered at intervals of 2 hours to analyze the asparaginase secreted by the medium, and the cell growth curve and asparaginase enzyme activity were measured. As a result, all of the recombinant strains did not show any signs of stress in the cells, and it was confirmed that the cell absorbance (OD _600nm ) was well above 100 and reached a very high concentration within 14 hours after the culturing. It was also confirmed that the enzyme activity gradually increased with the incubation time. The secreted asparaginase was recovered by using an anion exchange resin as in Example 6 above. As a result, it was confirmed that the recovered asparaginase was separated at a purity of 95% or more, and compared with the amount of the purified protein in the recombinant strain having the ss2629-secretory sequence (114 mg / L), R2-ss2629- and R3- , It was possible to obtain more protein (145 mg / L, 166 mg / L). As a result, it was confirmed that asparaginase could be overproduced using the secretion sequence using the ramp tag function.

2-Dimensional Electrophoresis was used to determine whether the asparaginase protein produced and purified from the recombinant Corynebacterium glutamicum strain was transformed by unintended side reactions during post-translational modification or separation and purification. Respectively.

Asparaginase protein produced and purified from the recombinant Corynebacterium glutamicum strain was subjected to a sample analysis by ProteomeTech, Korea through the procedure of Example 6. As a result, pXMJ19 / ssNCgl2629-asnB recombinant corn In the case of asparaginase produced from four bacterium glutamicum, it was confirmed that the protein was transformed to a lower level than the commercially available asparagine-derived asparaginase preparation (Fig. 9). As shown in the figure, the number of confirmed spots is 5, and the ratio of the most important one (no. 4) is about 68.2%. This result shows that the ratio of high purity and major spot is remarkably higher than that of commercially available protein preparation.

Spot Volume ratio (% Volume) Relative intensity against # 4 (%) One 1.21254 1.78 2 2.14667 3.15 3 8.09859 11.87 4 68.2028 100.00 5 11.4753 16.83

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Secretory production of L-asparaginase in recombinant          Corynebacterium <130> 40670636 <160> 57 <170> KoPatentin 3.0 <210> 1 <211> 22 <212> PRT <213> Corynebacterium ammoniagenes <400> 1 Met Lys Arg Met Lys Ser Leu Ala Ala Ala Leu Thr Val Ala Gly Ala   1 5 10 15 Met Leu Ala Ala Pro Val              20 <210> 2 <211> 27 <212> PRT <213> Corynebacterium glutamicum <400> 2 Met Phe Asn Asn Arg Ile Arg Thr Ala Ala Leu Ala Gly Ala Ile Ala   1 5 10 15 Ile Ser Thr Ala Ala Ser Gly Val Ala Ile Pro              20 25 <210> 3 <211> 29 <212> PRT <213> Paenibacillus sp. <400> 3 Met Gly Leu Lys Met Lys Lys Arg Ser Gly Lys Lys Ala Trp Met Leu   1 5 10 15 Leu Val Met Ser Leu Leu Ile              20 25 <210> 4 <211> 32 <212> PRT <213> Listeria monocytogenes <400> 4 Met Arg Lys Lys Arg Tyr Val Trp Leu Lys Ser Ile Leu Val Ala Ile   1 5 10 15 Leu Val Phe Gly Ser Gly Val Trp Ile Asn Thr Ser Asn Gly Thr Asn              20 25 30 <210> 5 <211> 26 <212> PRT <213> Corynebacterium glutamicum <400> 5 Met Lys Leu Leu Arg Arg Ile Ala Ala Pro Ala Ile Ala Leu Gly Ile   1 5 10 15 Ala Met Ser Thr Ile Val Thr Pro Ser Thr              20 25 <210> 6 <211> 23 <212> PRT <213> Corynebacterium glutamicum <400> 6 Met Lys Lys Leu Arg Phe Ala Thr Ile Ala Ala Ala Thr Val Ala Leu   1 5 10 15 Thr Ala Ser Leu Thr Pro Ser              20 <210> 7 <211> 21 <212> PRT <213> Corynebacterium glutamicum <400> 7 Met Arg Lys Phe Arg Asn Thr Ala Ile Ala Leu Val Ser Ala Ala Ala   1 5 10 15 Ile Thr Leu Gly Gly              20 <210> 8 <211> 28 <212> PRT <213> Corynebacterium glutamicum <400> 8 Met Arg Lys Gly Ile Ser Arg Val Leu Ser Val Ala Val Ala Ser Ser   1 5 10 15 Ile Gly Phe Gly Thr Val Leu Thr Gly Thr Gly Ile              20 25 <210> 9 <211> 34 <212> PRT <213> Corynebacterium glutamicum <400> 9 Met Ser Val Phe Thr Arg Ala Gly Glu Ala Ser Arg Lys Leu Val Ala   1 5 10 15 Leu Val Ala Leu Ala Ala Leu              20 25 30 Gly Thr         <210> 10 <211> 32 <212> PRT <213> Corynebacterium glutamicum <400> 10 Met Thr Ser Ser Phe Ser Arg Arg Gln Phe Leu Leu Gly Gly Leu Val   1 5 10 15 Leu Ala Gly Thr Gly Ala Val Ala Ala Cys Thr Ser Asp Pro Gly Pro              20 25 30 <210> 11 <211> 30 <212> PRT <213> Corynebacterium glutamicum <400> 11 Met Arg Ser Leu Met Pro Gln Leu Ser Arg Arg Gln Phe Leu Gln Thr   1 5 10 15 Thr Ala Val Thr Ala Gly Leu Ala Thr Phe Ala Gly Thr Pro              20 25 30 <210> 12 <211> 23 <212> PRT <213> Corynebacterium glutamicum <400> 12 Met Ala Gln Ile Ser Arg Arg His Phe Leu Ala Ala Ala Thr Val Ala   1 5 10 15 Gly Ala Gly Ala Thr Leu Ala              20 <210> 13 <211> 27 <212> PRT <213> Corynebacterium glutamicum <400> 13 Met Gln Ile Asn Arg Arg Gly Phe Leu Lys Ala Thr Thr Gly Leu Ala   1 5 10 15 Thr Ile Gly Ala Ala Ser Met Phe Met Pro Lys              20 25 <210> 14 <211> 37 <212> PRT <213> Corynebacterium glutamicum <400> 14 Met Val Asn Thr Leu Asn Ser Lys Thr Val Asn Val Pro Arg Phe Ala   1 5 10 15 Arg Gly Val Val Ala Ala Ala Leu Phe Phe Gly Ala Leu Val              20 25 30 Ser Leu Ala Pro Ser          35 <210> 15 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> gfpuv forward primer <400> 15 ccaaagctta tggcatgcct gcaggtcgac tc 32 <210> 16 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> gfpuv reverse primer <400> 16 ccagaattct tatttgtaca gctcatcc 28 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Rluc8_19 forward primer <400> 17 aaaaagctta tggcttccaa ggtgtacg 28 <210> 18 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Rluc8_19 reverse primer <400> 18 aaagaattct caatgatgat gatgatgatg gtcg 34 <210> 19 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Rluc8 (ecoRV) forward primer <400> 19 atagatatca tggcttccaa ggtgtacgac c 31 <210> 20 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> gfpuv (ecoRV) forward primer <400> 20 atagatatcg catgcctgca ggtcgactc 29 <210> 21 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> plac R (ecoRV) reverse primer <400> 21 atagatatca attaattctg tttcctgtgt gaaattgtta tcc 43 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> cspA forward primer <400> 22 atgaaacgca tgaaatcgct gg 22 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> cspA reverse primer <400> 23 tttttctgct gccgttgcc 19 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cspB forward primer <400> 24 atgtttaaca accgtatccg cactg 25 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> cspB reverse primer <400> 25 ggtttcctga gcgaatgctg g 21 <210> 26 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> psp forward primer <400> 26 atgggtttga agatgaagaa aagatcag 28 <210> 27 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> psp reverse primer <400> 27 atccgatgct gcgctgg 17 <210> 28 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> inlaid forward primer <400> 28 atgagaaaaa aacgatatgt atggttgaaa ag 32 <210> 29 <211> 23 <212> DNA <213> Artificial Sequence <220> Reverse primer <400> 29 aattgtagct gcctgagcat ttg 23 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NCgl0336 forward primer <400> 30 atgaagcttc ttcgccgcat c 21 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NCgl0336 reverse primer <400> 31 aattgtagct gcctgagcat ttg 23 <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NCgl0932 forward primer <400> 32 atgaagaaac tacgtttcgc cacc 24 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NCgl0932 reverse primer <400> 33 gaaatcctgt gcggaagctg ag 22 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NCgl1930 forward primer <400> 34 atgcgtaaat tccgcaacac tg 22 <210> 35 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NCgl1930 reverse primer <400> 35 agtttcgtct tcctgagcgg tag 23 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NCgl2101 forward primer <400> 36 atgcgtaaag gaatttcccg cg 22 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NCgl2101 reverse primer <400> 37 agagtcttga gctgctgcga tg 22 <210> 38 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NCgl2779 forward primer <400> 38 atgtccgtat ttacacgagc tgg 23 <210> 39 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NCgl2779 reverse primer <400> 39 acggtttgca gcttgtgc 18 <210> 40 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NCgl2865 forward primer <400> 40 atgacaagta gtttttcccg gcg 23 <210> 41 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> NCgl2865 reverse primer <400> 41 acctggtgcc gaggcag 17 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NCgl0801 forward primer <400> 42 atgcaaataa accgccgagg c 21 <210> 43 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NCgl0801 reverse primer <400> 43 tgctccaagg gcgttggc 18 <210> 44 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NCgl2185 forward primer <400> 44 atgccacagt taagcagacg c 21 <210> 45 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NCgl2185 reverse primer <400> 45 gcgttcttca gcgcgtgc 18 <210> 46 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NCgl2629 forward primer <400> 46 atggtgaaca cgttgaactc taaaacc 27 <210> 47 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> NCgl2629 reverse primer <400> 47 tggttcctgc gccaacg 17 <210> 48 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NCgl1331 forward primer <400> 48 atggctcaga tttctcgtcg tc 22 <210> 49 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> NCgl1331 reverse primer <400> 49 accggtaccc gcacatg 17 <210> 50 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Asp_19 forward primer <400> 50 cctaagctta tggagttttt caaaaagacg gc 32 <210> 51 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Asp_19 reverse primer <400> 51 actgaattct tagtactgat tgaagatctg ctgg 34 <210> 52 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> asp forward (ecoRV) primer <400> 52 atagatatca tggagttttt caaaaagacg gcacttg 37 <210> 53 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> plasc reverse (ecoRV) primer <400> 53 atagatatca attaattctg tttcctgtgt gaaattgtta tcc 43 <210> 54 <211> 117 <212> DNA <213> Artificial Sequence <220> <223> ramp tag R1-ss2629 <400> 54 gtaaatacac tcaatagtaa aacggtcaat gtacctcggt ttgcacgggg cgtagtagcc 60 gctgcgacag cgttattttt cggggcccta gtttcgcttg ccccctcggc gctagcg 117 <210> 55 <211> 117 <212> DNA <213> Artificial Sequence <220> <223> ramp tag R2-ss2629 <400> 55 gtcaacacac tgaattcgaa gaccgtaaac gtacctcgat ttgcgcgagg agtggtagca 60 gccgcgacag ctttattttt tggggcctta gtttcgctcg ctccgagtgc ccttgcc 117 <210> 56 <211> 117 <212> DNA <213> Artificial Sequence <220> <223> ramp tag R3-ss2629 <400> 56 gtaaatacgt taaacagtaa gacggtgaat gtaccgagat tcgcaagagg tgtggtagcg 60 gcagccacgg cgctattctt cggtgctctt gtaagtcttg caccctcggc cctagca 117 <210> 57 <211> 117 <212> DNA <213> Artificial Sequence <220> <223> ramp tag R4-ss2629 <400> 57 gtgaacacat tgaattcgaa aacggtgaat gtgccccgct ttgctagagg agtagttgcg 60 gccgcaacgg cactattctt cggggctcta gtgagtttag caccgagtgc cttagct 117

Claims (9)

(SEQ ID NO: 54), SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57 in the signal sequence, wherein the signal sequence comprises an asparaginase gene linked to a signal sequence consisting of Corynebacterium- Recombinant vector for tagged asparaginase expression and secretion increase.
The method according to claim 1,
Wherein the signal sequence is a signal sequence of a cell surface protein or secretory protein of Corynebacterium.
delete The method according to claim 1,
The Corynebacterium (Corynebacterium) is Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium ammoniagenes to Ness (Corynebacterium ammoniagenes), Corynebacterium Kasei (Corynebacterium casei ) and Corynebacterium efficiens. &lt; / RTI &gt;
The method according to claim 1,
The asparaginase gene is expressed by Escherichia coli ) or Erwinia carotovora ) derived recombinant vector.
delete delete A recombinant Corynebacterium strain transformed with the recombinant vector of any one of claims 1, 2, 4 and 5.
A method for producing asparaginase from Corynebacterium transformed with the recombinant vector of any one of claims 1, 2, 4 and 5.

Images