KR100672745B1 - Methods for enhancing a secretion efficiency of recombinant foreign protein in yeast expression system - Google Patents

Abstract

The present invention relates to a method for improving the secretion efficiency of recombinant foreign protein in a yeast expression system. More specifically, the method for improving the secretion efficiency of the foreign protein of the present invention is a recombinant foreign gene construct expressed under the influence of (i) a galactose-induced promoter, a secretion sequence and a galactose-induced promoter sequentially comprising a gene encoding the foreign protein. Transducing yeast into host yeast to prepare a transgenic yeast strain; And (ii) culturing the transgenic yeast strain under conditions in which the activity of the galactose inducing promoter is controlled. The method for improving the secretion efficiency of the foreign protein of the present invention by appropriately controlling the level of galactose acting as an inducer of galactose-induced promoter in cells, recombination by overexpression generated in the yeast expression system based on the conventional galactose-induced promoter By reducing the insoluble precipitation of the foreign protein, ultimately to improve the secretion efficiency of the recombinant foreign protein, it can be usefully used to improve the productivity of the recombinant foreign protein in the yeast expression system, and to lower the production cost.

Protein secretion, galactose-induced promoter, Saccharomyces cerevisiae, catabolism inhibition, catabolite repression, galactose transport, mixed carbon source injection

Description

Methods for enhancing a secretion efficiency of recombinant foreign protein in yeast expression system

1 is a conceptual diagram illustrating a process in which galactose in a medium passes through a yeast cell membrane and acts on an exogenous protein expression promoter, and a process in which glucose causes catabolite repression in a cell.

2 is a cleavage map of the expression vector MδLK8 of the recombinant LK8 protein used in the present invention.

3 is a graph comparing galactose consumption rate and cell growth rate with time when galactose was supplied as the only carbon source in two host yeasts having the same genetic background except for the Gal2 gene.

4A is When the exogenous gene is expressed in the transformed yeast A, it is a photograph showing the result of Western blot analysis of the total intracellular exogenous protein according to the expression induction time, Figure 4b is the intracellular water-soluble foreign protein in the transformed yeast A It is a photograph showing the result of Western blot analysis.

Figure 5 is a photograph showing the results of Western blot analysis of the total intracellular foreign protein according to the expression induction time, when the foreign gene is expressed in the transformed yeast B. Compared with yeast A with GAL2 gene as a host cell, the amount of protein accumulated due to intracellular insoluble decreases with time.

6 is transformation When yeasts A and B are cultured and the foreign genes are expressed, it is a graph comparing the amount of recombinant foreign protein secreted into the medium according to expression induction time.

Figure 7 is, by the time the exogenous protein expressed galactose in yeast protein disulfide isomerase is a cleavage map of the expression vector that allows pMPDI1 (protein disulfide isomerase, PDI1) can be co-expressed.

The present invention relates to a method for improving secretion efficiency of recombinant foreign protein in a yeast expression system.

Mass production of foreign proteins using microorganisms is a key technology field in the protein pharmaceutical manufacturing industry. Expression-derived proteins can be expressed intracellularly or secreted extracellularly for recovery and purification after expression of the foreign protein. In the case of intracellular expression, in many cases, the protein is over-expressed, resulting in the inactivation of the inactivated state in the cell, and in many cases, the productivity is reduced, such as the difficulty of breaking down the hard microorganisms and the difficulty of the purification process to separate from the numerous proteins present in the cell. Provide factors. On the other hand, if the produced protein is secreted extracellularly, it is easy to avoid the difficulties of the former case. In addition, the extracellular secretion of the protein is possible only when the protein is folded and modified correctly after transcription, and thus there is an advantage of obtaining a soluble protein having an accurate tertiary structure in an activated form when producing a therapeutic protein. Therefore, the extracellular secretion of protein is superior to the intracellular accumulation system of recombinant foreign protein in terms of protein production yield as well as protein quality control.

In the recombinant protein expression system using a strong promoter, however, extracellular secretion is often significantly lower than that of intracellular accumulation. To overcome this point, many studies have been conducted to increase the extracellular secretion yield. Most studies have focused directly on optimizing the secretion sequence of proteins or finding new powerful secretion sequences (Nucleic Acids Res Suppl., 2003 (3): 261-2; Biochem Cell Biol. 1993, 71: 401-5). .

On the other hand, by using molecular biological techniques, the overexpression of chaperones, which help to fold and soluble in proteins, facilitates the folding of proteins, which are the initial rate step, and secretion into the cell when overexpression of recombinant foreign protein. Many studies have been carried out to prevent insoluble sedimentation and ultimately increase secretion efficiency (Robinson and Wittrup, Biotech. Prog ., 11: 171, 1995; Robinson et al., Biotechnology , 12: 381-384, 1994; Wulfing and Plukthun, Mol.Microbiol . 12 (5): 685-692, 1995).

Galactose promoters commonly used to induce the expression of foreign recombinant proteins in yeast are powerful inducible promoters that use galactose as an inducer. Such strong expression-inducing action of the protein is advantageous to increase the intracellular accumulation or expression of the foreign protein, but when secreting the protein of interest to the extracellular secretion, intracellular insoluble sedimentation occurs in the early stage of secretion and secretion efficiency is increased. Rather, they can fall or paralyze the function of the cell. This phenomenon is often observed in recombinant expression systems such as E. coli or yeast expression systems, and there have been attempts to lower the culture temperature to overcome this problem (Baneyx F., Curr . Opin . Biotechnol ., 10: 411-421, 1999; George Georgiou and Pascal, Curr . Opin . Biotechnol ., 7: 190-197, 1996).

However, when the incubation temperature is lowered as described above, the incubation time is increased, the amount of absolute protein expression is reduced, and the production cost of the recombinant foreign protein is increased. Therefore, there is an urgent need for the development of a method for improving the secretion efficiency of recombinant foreign proteins by controlling the activity of galactose-induced promoters without lowering the culture temperature.

Accordingly, the present inventors have focused on the fact that the activity of galactose-induced promoters can be regulated by regulating the host's availability of galactose, thereby using a mutant strain lacking the galactose permease gene associated with galactose uptake as a host or catabolic product. The present invention was completed by confirming that the secretion efficiency of recombinant foreign protein is improved when culturing the transformed yeast in a fed-batch culture in which glucose inducing inhibition is injected with galactose at an appropriate ratio.

An object of the present invention is to provide a method for improving the extracellular secretion efficiency of recombinant foreign protein in a yeast expression system through genetic modification of host yeast or environmental changes in culture.

Another object of the present invention is to provide a fed-batch culture method for improving the secretion efficiency of recombinant foreign protein.

In order to achieve the above object, the present invention is (i) transducing a recombinant foreign gene construct expressed under the influence of a galactose inducing promoter comprising a gene encoding a galactose inducing promoter, a secretion sequence and a foreign protein into a host yeast. To prepare a transgenic yeast strain; And (ii) culturing the transgenic yeast strain under conditions in which the activity of the galactose-induced promoter is controlled.

In addition, the present invention comprises the steps of (i) preparing a recombinant foreign gene construct that is expressed under the influence of a galactose induction promoter sequentially comprising a gene encoding a galactose induction promoter, secretion sequences and foreign proteins; (Ii) transducing the recombinant foreign gene construct into host yeast to prepare a transgenic yeast strain; And (iii) culturing the transgenic yeast strain by controlling the ratio of galactose and glucose, thereby providing a fed-batch culture method of recombinant yeast for improving secretion of recombinant foreign protein.

In addition, the present invention (i) the protein expressed under the influence of the recombinant foreign gene construct and galactose-induced promoter, which is expressed under the influence of the galactose-induced promoter sequentially comprising a galactose-induced promoter, secretion sequence and a gene encoding a foreign protein Preparing a transgenic yeast strain by transducing a gene construct encoding a protein disulfide isomerase into a host yeast; And (ii) culturing the transgenic yeast strain under conditions in which the activity of the galactose-induced promoter is controlled.

Hereinafter, the present invention will be described in more detail.

The method for improving the secretion efficiency of the foreign protein of the present invention comprises (i) a host yeast with a recombinant foreign gene construct expressed under the influence of a galactose inducing promoter, a secretion sequence, and a gene encoding the foreign protein. Transforming to prepare a transformed yeast strain; And (ii) culturing the transformed yeast strain under conditions in which the activity of the galactose inducing promoter is controlled.

At this time, the step (iii) is not particularly limited thereto, but is preferably performed by inserting the recombinant foreign gene construct into the chromosome of the host yeast or by inserting it into a vector, and the galactose inducing promoter is particularly limited thereto. but are not, GAL1 promoter, GAL7 promoter, the S. cerevisiae GAL10 promoter described by SEQ ID NO: 3 in S. cerevisiae is described in SEQ ID NO 2 of S. cerevisiae is described in SEQ ID NO: 1 Or a combination or fusion promoter of the above promoters, more preferably GAL1 , and the secretion sequence is not particularly limited thereto, but the MATα secretion sequence described in SEQ ID NO: 4, S. K1 lethal toxin secretion sequence of cerevisiae (killer toxin signal, Brown, JL et al, the K1 killer toxin:. molecular and genetic applications to secretion and cell surface assembly in:.. Johnston, JR Molecular Genetics of Yeast- a practical approach the practical approach series, 1994, p. 217-265; Tokunaga, M. et al., Biochem.Res . Commun ., 144: 613-619, 1987), invertase of S. cerevisiae described in SEQ ID NO: 6 ) Secretion sequence (JP-A-1985-041488), lethal toxin secretion sequence of Kluyveromyces lactis described in SEQ ID NO: 7 (Sugisaki, Y. et al., Eur. J. Biochem ., 141: 241-245, 1984 ), SEQ ID NO lethal toxin minutes of Pichia acaciae described in 8 secretary (U.S. Patent No. 6,1,07,057 No.), lethal toxin secretion sequence of Hanseniaspora uvarum (Radler, F. et al , Arch Microbiol, 154 (2):... 175-178, 1990; Schmitt, MJ et al , J. Virol. , 68 (3): 1765-1772, 1994, or the lethal toxin secretion sequence of Pichia (Hansenular) anomala , preferably any of the secretory sequences that can be used in the yeast expression system. In addition, the secretory sequence may further include a precursor peptide sequence for improving secretion efficiency, and furthermore, such as KEX1 (for killer expression 1) or KEX2. The recognition site of secretion sequence peptidase may be additionally included (US Patent No. 4,929,553). An example of the recognition site of the secretion sequence peptidase is Pro-Met-Tyr.

In addition, in one embodiment of the present invention, the condition in which the activity of the galactose-induced promoter is regulated in step (ii) is preferably one in which the intracellular transport rate of galactose is reduced. At this time, the conditions in which the intracellular transport rate of galactose is reduced are not particularly limited thereto, but the strain of the galactose permease gene or the modified strain from which the galactose permease gene has been artificially removed by the host yeast of step (iii). It is preferable that the conditions for transducing a recombinant foreign gene construct expressed under the influence of a galactose inducing promoter and culturing the transduced strain in a galactose-containing medium, wherein the galactose permease defective strain is particularly limited thereto. However, it is preferred that it is Saccharomyces cerevisiae BJ3501 (ATCC 208280).

In addition, in another preferred embodiment of the present invention, the conditions under which the activity of the galactose-induced promoter is regulated in step (ii) are simultaneously injected with glucose inducing medium to cause catabolite repression of galactose and galactose-induced promoters. Condition. In this case, the galactose and glucose are not particularly limited thereto, but are preferably injected by oil culture, and more preferably in a ratio of 8: 1 to 1: 4.

In another preferred embodiment of the present invention, the conditions in which the intracellular transport rate of galactose is reduced are determined in the host yeast of step (iii), or the modified strain from which the galactose permease gene has been artificially removed. A recombinant foreign gene construct expressed under the influence of a galactose inducing promoter is transduced, and the transduced strain is cultured by injecting galactose and glucose in a fed-batch fashion.

In addition, the fed-batch cultivation method of recombinant yeast for improving secretion of recombinant foreign protein of the present invention is expressed under the influence of (i) a galactose-induced promoter, which includes a galactose-induced promoter, a secretion sequence and a gene encoding a foreign protein. Preparing a transgenic yeast strain by transforming the host yeast with the recombinant foreign gene construct; And (ii) culturing the transformed yeast strain by controlling the ratio of galactose and glucose.

At this time, the host yeast of step (iii) is not particularly limited thereto, but is preferably a strain of galactose permease gene deletion or a modified strain from which the galactose permease gene is artificially removed, and the galactose and glucose are particularly limited thereto. It is preferred, however, to inject at a ratio of 8: 1 to 1: 4.

In addition, the method for improving the secretion of foreign proteins of the present invention (i) a recombinant foreign gene construct expressed under the influence of galactose induction promoter, secretion sequence and galactose induction promoter sequentially comprising a gene encoding the foreign protein and Preparing a transformed yeast strain by transforming the host yeast with a gene construct encoding a protein disulfide isomerase expressed under the influence of a galactose inducing promoter; And (ii) culturing the transformed yeast strain under conditions in which the activity of the galactose inducing promoter is controlled.

At this time, the conditions in which the intracellular transport rate of galactose is reduced are not particularly limited thereto, but galactose is modified into galactose permease gene-deficient strains or modified strains in which galactose permease genes have been artificially removed by the host yeast of step (iii). Inducing a transgenic recombinant construct expressed under the influence of an inducible promoter and inducing catabolite repression of galactose and galactose inducing promoter in conditions or cultures in which the transduced strain is cultured in galactose containing media It is preferable that the conditions of injecting glucose at the same time, and the galactose permease gene deletion strain is not particularly limited, but Saccharomyces cerevisiae BJ3501 (ATCC 208280).

In one embodiment of the present invention, the gene encoding the protein disulfide isomerase is not particularly limited thereto, but PDI1 (Tachikawa, H. et al. S. cerevisiae described in SEQ ID NO: 9) . , J. Biochem., 110 (2): 306-313, 1991) , PDI of Conus textile described in SEQ ID NO: 10 (US Patent Publication No. US2004 / 0203132), PDI of C. elegans described in SEQ ID NO: 11 (Page , AP , DNA Cell Biol., 16 (11): 1335-1343, 1997), human pancreatic PDI gene Desilva, MG et al ., DNA Cell Biol ., 15 (1): 9-16, set forth in SEQ ID NO: 12 , 1997), PDI of Aspergillus oryzae, the fungus described in SEQ ID NO: 13 (International Publication No. WO95 / 00636), PDI of Candida boidinii described in SEQ ID NO: 14 (US Pat. No. 5,965,426), or SEQ ID NO: 15 of Humicola insolens PDI (U.S. Patent No. 5700659 No.) is desirable, and the protein disulfide isomerase effect Gene construct encoding a (protein disulfide isomerase) teuneun not particularly limited to, preferably in the pMPDI having a cleavage map set forth in FIG.

In yeast expression system, galactose-induced promoters having strong expression intensity are frequently used. The galactose inducing promoter is activated when galactose used as an inducing substance is present in the cell, thereby mass-expressing a gene under the influence of the promoter. Because of this inducibility, it is used as a useful tool for expressing foreign recombinant proteins in yeast. However, as described above, the present inventors observed intracellular insoluble precipitation even before the foreign protein was secreted in the early stage of expression (see FIG. 4A). Therefore, in order to overcome this and ultimately improve the extracellular secretion of foreign proteins, the present inventors have concluded that the overactivation of the galactose-induced promoter due to the excess of galactose in the cells is the main cause of intracellular insoluble precipitation before secretion of the recombinant foreign protein. With the focus on, it was confirmed that the secretion efficiency of the recombinant foreign protein was improved by controlling the intracellular inflow rate of galactose used as the expression inducing agent of the galactose inducing promoter.

First, the analysis of the intracellular absorption pathway of galactose shows that intracellular transport of galactose is possible through several pathways. The most preferred transport pathway for galactose is expression of galactose in the presence of galactose as a component of galactose operon. It is the most affinity galactose permease (Gal2). However, the host without galactose permease (Gal2) does not completely block the transport of galactose, but has a relatively low affinity for galactose in a secondary way, but HXT1, HXT9, HXT11, HXT14, etc. Since hexose transporters can transport galactose into cells (Wieczorke R. et al ., FEBS . Lett . 464 (3): 123-128, 1999), GAL promoter activation Minimal galactose movements are possible (see FIG. 1). FIG. 1 is a conceptual diagram illustrating a process in which galactose in a medium passes through a yeast cell membrane and acts on a foreign protein expression inducing promoter, and a process in which glucose causes catabolite repression in a cell.

Accordingly, the present inventors have determined that when the yeast lacking a gene ( GAL2 ) encoding Gal2, which is a major transporter of galactose, is used as a host, the intracellular transport of galactose can be minimized. First, the GAL2 gene is deleted. Transformed by introducing a recombinant foreign gene construct that expresses a foreign gene under the influence of a galactose-induced promoter using a yeast strain (S accharomyces cerevisiae BJ3501 (ATCC 208280), hereinafter referred to as 'host yeast B') Yeast (hereinafter, referred to as 'transformed yeast B'), and the transduced yeast are cultured in a galactose-containing medium, so that the gene is not deleted normal yeast ( Saccharomyces cerevisiae 2805, hereinafter, 'host yeast A' Secretion efficiency of the transformed yeast (hereinafter referred to as 'transformation yeast A') and the recombinant foreign protein was compared (see FIG. 3). As shown in Figure 3, when hayeoteul except GAL2 gene were cultured in a medium containing the genetic background, the same host yeast A and B each of galactose as the sole carbon source, was measured galactose nongdoeul in the culture medium over time and comparing, GAL2 According to the presence or absence of the gene, it was confirmed that the intracellular inflow rate of galactose showed a big difference.

Furthermore, when host yeast B, which has a low intracellular inflow rate of galactose due to the inability of the GAL2 gene, is used as a host for exogenous protein expression, intracellular insoluble precipitation due to overexpression of foreign protein under the action of galactose-induced promoter Yeast A can be confirmed by Western blot analysis using a foreign protein antibody, which is relatively reduced compared to when expressed using a host for foreign protein expression (see FIGS. 4A and 4B) (see FIG. 5). In addition, it was confirmed that the extracellular secretion and secretion efficiency of the foreign protein, which is the ultimate result to be provided by the present invention, together with the reduction of the insoluble precipitate in the cell under actual culture conditions, was increased through HPLC analysis of the secreted foreign protein in the culture medium (Fig. 6). In one embodiment of the present invention, in order to reduce the intracellular influx rate of the galactose, a yeast strain lacking the galactose permease gene was used as a host. Alternatively, antisense, siRNA, antibodies, antagonists and the like that inhibit the function of the gene may be used to inhibit the function of the gene.

In order to appropriately control the availability of intracellular galactose, the present inventors have identified a catabolism with galactose, an inducer of a galactose-induced promoter (when glucose, the primary preferred sugar of a cell, is present in the medium, glucose is a carbon source other than glucose). Inhibiting the expression of genes involved in the consumption of nutrients) by simultaneously injecting glucose into the fermentation medium so that the host yeast consumes both sugars at the same time. A strategy in which protein overexpression is appropriately suppressed was employed. 1 is a conceptual diagram illustrating the mutually opposite actions such as induction and inhibition of galactose and glucose in the cells (G.-D. Entian and H.-J. Schuller, Glucose Repression in Yeast. Yeast Sugar Metabolism , FK Zimmermann, K.-D. Entian., Eds.pp. 409-434.Technomic Publishing AG, Bassel, Switzerland, 1997). At this time, the galactose-induced promoter is completely inhibited by Klucos by injecting a mixed carbon source through a fed-batch culture method in which the glucose concentration in the culture medium is maintained at 0.1 g / L or less. To prevent it. The inventors have named the above method as a mixed-carbon source feeding strategy. The present invention provides a method for artificially controlling the amount of foreign protein secretion in each case after culturing with various ratios of glucose / galactose-containing milk medium, and also comparing the amount of foreign protein secretion at each rate to provide a host with different galactose transport capacity. Optimal glucose / galactose ratios required for exogenous protein secretion of yeast were identified (see Table 1 and Table 2). When the mixed carbon source injection method designed by the present invention is applied to host yeast A and host yeast B, respectively, compared with the general expression induction method using only galactose as a single carbon source, the production yield of foreign protein per unit cell and foreign per unit inducing agent Protein yields increased 105% (Table 1) and 85% (Table 2), respectively. In addition, when the transgenic yeasts A and B were cultured by applying a mixed carbon source medium injection method, it was found that different optimal glucose / galactose concentrations existed. In the case of transgenic yeast A, which has a relatively high rate of galactose inflow and consumption, the glucose / galactose ratio of the mixed carbon source for optimal secretion of foreign protein was about 1: 1 and the galactose transport capacity of the cell was not due to the galactose permease not working. In the case of the reduced transgenic yeast B, the optimal ratio of mixed carbon source (glucose / galactose) was about 2: 3, and the ratio of galactose in the medium was relatively higher than that of transgenic yeast A (Table 1 and Table 2). Reference). This results indirectly prove that the galactose transport capacity and the effect of the mixed carbon source on the foreign protein expression in the foreign protein expression system using the galactose-induced promoter are in good agreement with each other as intended.

On the other hand, the inventors of the present invention to the host cell with a foreign gene construct that is expressed under the influence of the galactose-induced promoter gene ( PDI1 ) encoding a protein disulfide isomerase known to help fold the protein When transduced, it was confirmed that the protein secretion efficiency is improved.

First, the host with a saccharide which is described in SEQ ID NO: 9 to prepare a expression vector pMPDI1 containing the gene (PDI1) of my process the protein disulfide isomerase of three Levy jiae, and the expression vector with the foreign gene construct Yeast A and B were each transduced. Subsequently, the transduced yeast was cultured in a galactose-containing medium to measure protein secretion efficiency (see Table 3). As a result, by using host yeast A case where transduced with the exogenous gene construct and pMPDI1, it was secreted efficiency is improved to 30% with respect to the control group (host yeast A is not introduced into the PDI1 gene), using host yeast B In this case, it was found that the secretion efficiency improved by 72% with respect to the control group.

As described above, the method for improving the secretion efficiency of the foreign protein of the present invention in the yeast expression system based on the conventional galactose-induced promoter by appropriately adjusting the level of galactose acting as an inducer of the galactose-induced promoter in cells It is possible to reduce the insoluble precipitation of recombinant foreign protein due to overexpression, which can ultimately improve the secretion efficiency of recombinant foreign protein, which is useful for improving the productivity of recombinant foreign protein in yeast expression system and lowering the production cost. Can be used.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Comparison of Galactose Intracellular Transport Capacity of Host Yeast A and Host Yeast B

Host yeast A ( S. cerevisiae 2805, Genotype: MATα pep4 :: HIS3 prb1-Δ1.6R his3-Δ200 ura3-52 GAL2 can1, Sohn, JH et al . Proc. Biochem ., 30: 653-660, 1995; Kim , TH et al . Biotechnol. Lett . 24: 279-286, 2002) and host yeast B ( S. cerevisiae BJ3501, Genotype: MATα pep4 :: HIS3 prb1-Δ1.6R his3-Δ200 ura3-52 gal2 can1 , ATCC 208280 , USA) was linearly streaked onto a plate of YPD agar, and then left to stand at 30 ° C. for about 18 hours to separate colonies. Colonies of A and B were inoculated in different YPG [yeast extract 1% (w / v), peptone 2% (w / v), galactose 2% (w / v)] liquid medium, and then incubated at about 80 ° C. in a thermostat. Time shake cultures. In order to measure galactose consumption over time, the remaining sugars of the medium were quantified by the DNS method (3,5-dinitrosalicylic acid assay) (Mohun, AF and Cook, IJ, J. Clin . Pathol ., 15: 169-). 180, 1962, FIG. 3). 3 is a graph comparing galactose consumption rate and cell growth rate with time when galactose was supplied as the only carbon source in two host yeasts having the same genetic background except for the deletion of the GAL2 gene. As shown in Figure 3, there GAL2 gene is the maximum consumption rate and cell growth rate of galactose units per hour in a yeast deficient host B can check the reduced compared to the host yeast GAL2 gene A, which is operating normally.

Example 2 Transformation Yeast Preparation Producing Foreign Proteins Using Host Yeasts A and B as Hosts

<Example 2-1> Preparation of exogenous protein secretion transformed yeast S. cerevisiae 2805 / MδLK8 using host yeast A

pMBRI-LK8 expression vector used by the present inventors to produce recombinant LK8 in Pichia pastoris to separate the α-factor secretion signal and the LK8 cDNA together (Korean Patent Application No. 2004 -0069840). This document is incorporated herein by reference. The pMBRI-LK8 was treated with Eco R I restriction enzyme for 7 hours and washed using a PCR purification kit (Purification Kit, Qiagen, USA). Subsequently, after treatment with Bam HI restriction enzyme for 7 hours, DNA was separated by electrophoresis and the α-factor secretion signal described in SEQ ID NO: 4 using a gel extraction kit (Qiagen, USA) and SEQ ID NO: 16 are described. DNA fragments with the resulting LK8 cDNA sequences were obtained. The obtained DNA fragment was inserted between the GAL1 promoter and the CYC1 terminator of the p426GAL1 (ATCC 87833, USA) vector to prepare an expression vector pMCLK8 (6.9 kb) which can be used to produce recombinant LK8 in yeast. The expression vector has a GAL1 promoter to induce protein expression by galactose. URA3 markers were used for selection in selection medium after transformation of yeast. Then, in order to insert the expression cassette consisting of the α-secretion sequence and the cDNA of LK8 into the chromosome of the yeast, the δ sequence and insertion so that the desired gene can be inserted into the δ sequence, which is one of the transition elements present in the chromosome of the yeast The pδneo vector (Lee, FW and Da Silva, NA, Appl. Microbiol. Biotechnol. , 48: 339, 1997) containing the neomycin resistance gene ( neo ) for selection of the isolated vector was inserted by the following method: The LK8 expression cassette was isolated using Sac I and Kpn I restriction enzymes that cut both ends of the GAL1 promoter and the CYC1 terminator, respectively, in the pMCLK8 vector. At this time, the Sal I restriction site exists in the sequence of the LK8 expression cassette and the pδneo vector, and the Sal I restriction enzyme site in the pδneo vector is necessary for insertion into the yeast chromosome. Takara, Japan) was used to remove Sal I restriction enzyme sites present in the LK8 expression cassette. On the other hand, in the pδneo vectors Kpn because I restriction site is not present, the DNA smoothing kit pδneo Xba I restriction site and the LK8 expression Kpn I all of the restriction site blunt-end (blunt end) of the cassette separation of the vector using made. Subsequently, the smoothed LK8 expression cassette and the pδneo vector were ligated with a ligase to prepare a recombinant vector, which was named MδLK8 recombinant expression vector (FIG. 2). 2 is a cleavage map of the expression vector MδLK8 of the recombinant LK8 protein used in the present invention.

Subsequently, Saccharomyces cerevisiae 2805 (Sohn, JH et al . Proc. Biochem .) Was prepared using the MδLK8 recombinant expression vector prepared above using an alkaline cationic yeast transformation kit (Q-BIOgene, Canada). , 30: 653-660, 1995; Kim, TH et al ., Biotechnol. Lett ., 24: 279-286, 2002). Yeast nitrogen base without amino acid 0.67% (w / v) (Difco, USA), yeast synthetic drop-out medium supplement without uracil 0.192% (w / v) (Difco, USA). The yeast transformed with the selected MδLK8 recombinant expression vector was obtained from YPD plates containing G418 sulfate antibiotics [2% (w / v) peptone, 1% (w / v) yeast extract, 2% (w / v). ) glucose and 2% (w / v) agar]. At this time, the concentration of the antibiotic G418 sulfate was adjusted to 5 g / L, 10 g / L and 15 g / L, respectively, to select a strong strain resistant to antibiotics in the transformed yeast strain. This was named Saccharomyces cerevisiae 2805 / MδLK8. The strain is hereinafter abbreviated as transformed yeast A for convenience.

Example 2-2 Foreign Protein Secreted Transformed Yeast Using Host Yeast B S. cerevisiae  Manufacture BJ3501 / MδLK8

Except for the fact that the GAL2 gene was deleted by the recombinant expression vector MδLK8 prepared in Example 2-1, Saccharomyces cerevisiae BJ3501 (ATCC 208280, USA), the same yeast strain as Saccharomyces cerevisiae 2805, was used. Transformation was performed in the same manner as. Through colony screening, clones with the highest secretion efficiency were selected and finally named Saccahromyces cerevisiae BJ3501 / MδLK8 # 36, and deposited on January 13, 2004 at the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10582BP). The transformed strain is hereinafter abbreviated as transformed yeast B for convenience.

Example 3 Comparison of Foreign Protein Secreting Capacity of Transformed Yeasts A and B in Fed-Batch Culture Using Galactose as Carbon Source

When the transformed yeasts A and B prepared in Example 2 were cultured in a fed-batch using galactose, the following analysis was performed to compare the secretion efficiency of foreign proteins: First, the transformed strain was used as a seed. YPD (yeast extract 1% (w / v) with 2% glucose (w / v) added to achieve adequate cell mass and activity (OD ₆₀₀ = 0.8 to 1.2 when diluted 20-fold) , peptone 2% (w / v)] in the culture medium was carried out at 1 to 3 vvm of air supply, agitation speed of 200 to 1000 rpm. After the seed culture was carried out in the YPD medium, seed culture solution was inoculated into the initial starting medium. First, the batch culture step is an adaptation step of galactose used as a substance for inducing cell growth and expression of foreign protein LK8, and inoculates at least 1% (v / v) of the culture medium and quantitatively uses the glucose and galactose as carbon sources. The period of galactose adaptation was increased. The initial culture medium used was glucose 2% (w / v), galactose 3% (w / v), yeast extract 4% (w / v), casamino acid (casamino acid) 0.5% (w / v), Uracil 0.5 g / L and histidine 0.5 g / L.

When the carbon source added to the initial medium in the batch culture step is depleted, the respiratory action (oxygen consumption) of the cell is lowered, which can be seen as a sudden increase in dissolved oxygen level by the dissolved oxygen probe installed in the incubator. From this point, fed-batch cultivation is performed by adding a medium containing galactose as a single carbon source in a DO-stat manner. The fed-batch culture step is to increase the concentration of cells and maintain the expression induction period of the foreign protein more continuously to improve the productivity of the foreign protein.

Specifically, a milky medium consisting of galactose 50% (w / v), yeast extract 30 g / L, peptone 20 g / L, uracil 1 g / L and histidine 2 g / L was used (milk medium I). At this time, galactose was supplied at a rate of 1 ml / hr to 30 ml / hr to induce mass secretion of the foreign protein LK8 so that the amount of galactose remaining in the medium was maintained at 5% (w / v) or less. The galactose supply method used a method of maintaining the amount of remaining galactose at an appropriate value by measuring the concentration of remaining galactose in the culture medium and adding galactose to an appropriate value based on this. By controlling the addition rate of the galactose it was possible to continuously increase the expression and secretion of LK8 while maintaining the concentration of galactose remaining in the fermentation tank below 5% (w / v) during the fermentation period.

In the fed-batch culture method, the expression and secretion patterns of the foreign proteins according to the galactose transport capacity of the yeast were compared to the transformed yeasts A and B prepared in Example 2. First, western blot analysis using anti-LK8 antibody was performed on the culture samples collected according to the elapsed time intervals in both cases to compare the distribution of foreign proteins accumulated in the cells. Each transgenic yeast sampled according to the elapsed time of culture was adjusted to the same amount, placed in the same amount of sample buffer, and cut and electrophoresed with SDS-polyacrylamide gel to develop intracellular proteins. The developed protein was transferred to nitrocellulose membrane. At this time, blotting was performed by adding a nitrocellulose membrane to a solution containing PBS buffer containing 0.1% (v / v) Tween 20 ^® and 5% (w / v) skim milk powder, and then stirring at room temperature for 2 hours. Stirred slightly. Subsequently, rabbit anti-LK8 antibody (rabbit anti-LK8 Ab) was added to the same solution as above, and gently stirred at room temperature for 1 hour, followed by 5 times with PBS buffer containing 0.1% (v / v) of Tween 20. Washed repeatedly. Anti-rabbit IgG-HRP (anti rabbit IgG-horseradish peroxidase, Sigma, USA) was then added to PBS buffer with 0.1% (v / v) Tween 20 and 5% (w / v) skim milk powder. The solution was added to the solution, and the treated nitrocellulose membrane was immersed in a solution to which the anti-rabbit IgG-HRP was added and gently stirred at room temperature for 1 hour. Later, 0.1% (v / v) detection solution to Tween 20 ^® are added to the washed repeatedly 5 times with PBS buffer, the nitrocellulose was taken out film chemiluminescent detection kit (SuperSignal ^TM West Pico kit, Pierce, USA) After the addition of the induced luminescence reaction, the light emitting nitrocellulose film was fixed to the photosensitive film, and the degree of shadowing of the droplets on the film was examined (FIGS. 4A, 4B and 5). 4A When the exogenous gene is expressed in the transformed yeast A, it is a photograph showing the result of Western blot analysis of the total intracellular exogenous protein according to the expression induction time, Figure 4b is the intracellular soluble exogenous protein in the transformed yeast A Figure 5 is a picture showing the results of Western blot analysis, Figure 5 is a picture showing the results of Western blot analysis of the total intracellular foreign protein according to the expression induction time, when the foreign gene is expressed in the transformed yeast B. As shown in FIG. 4A, when the GAL2 gene is normally operated and the galactose transport capacity is normal, it can be seen that the intracellular foreign protein accumulates as the culture culture inducing foreign gene expression continues. However, as shown in FIG. 4B, Western blot analysis of only intracellular soluble proteins revealed that the foreign protein is present in most insoluble proteins rather than soluble proteins over time of expression. And it was found. On the other hand, as shown in Figure 5, the transformed yeast B is deficient in the GAL2 gene galactose transport capacity was found that the intracellular accumulation of the foreign protein decreases with the expression time.

In order to confirm whether the result of FIG. 5 is due to the improvement of secretion efficiency of the foreign protein in the transformed yeast B lacking the GAL2 gene, or whether the overall protein expression level is reduced, it is prepared in Example 2 One transgenic yeast A and B were subjected to fed-batch culture under the same conditions, and high performance liquid chromatography (HPLC) analysis of the culture was performed to quantify the secreted foreign protein: first, the culture was centrifuged, The supernatant was filtered on a 0.2 μm filter. Subsequently, 100 μl of the filtered sample was measured for absorbance at a wavelength of 214 nm in HPLC. As a developing solvent, an acetonitrile containing 1% (w / v) trifluoroacetic acid (TCA) and water containing 1% (w / v) TCA were used as a mobile phase in a reverse phase separation column (Vydac ^TM c18 column). , Grace Vydac, USA) was used to isolate the foreign protein LK8. At this time, the composition of the mobile phase gave the gradient of the initial acetonitrile starting from 25% (v / v) to 40% (v / v). Accordingly, the hydrophobic difference was used to separate pure foreign protein of a single peak. Could. In addition, high purity purified foreign protein was measured by quantitative analysis of bicinchonic acid (BCA), and a calibration curve for the integral area and foreign protein concentration of HPLC was obtained. Using the calibration curve, the culture medium was analyzed by HPLC to obtain an integrated area, and at the same time, the aliphatic protein in the culture medium could be quantitatively analyzed by assigning it to a calibration curve using a standard sample (LK8) (FIG. 6). Figure 6 Transformation When yeasts A and B are cultured and the foreign genes are expressed, it is a graph comparing the amount of recombinant foreign protein secreted into the medium according to expression induction time. At this time, the closed ring (-●-) represents the extracellular secretion of the recombinant foreign protein in transformed yeast A ( S. cerevisiae 2805 / MδLK8), and the open rectangle (-□-) represents the transformed yeast B ( S. cerevisiae BJ3501). / Extracellular secretion amount of the recombinant foreign protein in MδLK8), respectively. As shown in FIG. 6, the extracellular secretion in the transformed yeast B with reduced galactose transport was about 2 times higher than that of the transformed yeast A that did not.

Example 4 Inhibition of Galactose Induced Promoters by Mixed-carbon Source Feeding in Transformed Yeast A and Comparison of Foreign Protein Secretion Yields

After inoculating the transformed yeast A prepared in Example 2-1 stored at −70 ° C. at 5 to 10% (v / v) in 10 ml of YPD medium for about 24 hours at 30 ° C. and 180 rpm in a stirred incubator. One step spawn culture. It was passaged in 200 ml of YPD medium and cultured in two stages for 24 hours under the same conditions. Yeast extract 4% (w / v), casamino acid 3% (w / v), histidine 0.05% (w / v), uracil 0.05% (w / v), glucose 2% (w / v) , Was carried out in a batch culture medium consisting of galactose 3% (w / v), the culture conditions were adjusted to a culture temperature 30 ℃, stirring speed 600 rpm, pH 5.0. Control of the batch culture was such that the speed of the stirrer was changed depending on the dissolved oxygen amount. In the latter part of the batch culture, dissolved oxygen is reduced due to the vigorous respiration of the cells, so to prevent this, the dissolved oxygen is maintained at 40% or more of the maximum dissolved oxygen by controlling the physical conditions of the air supply and the stirring speed until the end of the batch culture. . The oil price was followed by yeast extract (Difco, USA) 3% (w / v), peptone (Difco, USA) 2% (w / v), histidine (Sigma, USA) 0.2% (w / v), uracil ( Sigma, USA) 0.1% (w / v) of the culture medium fed to the galactose 50% (w / v) galactose, the control group of glucose and galactose ratio 1: 4 in the experimental group 10% glucose (w / v) ) And galactose 40% (w / v), glucose and galactose ratio 1: 1, glucose 25% (w / v) and galactose 25% (w / v), glucose and galactose ratio 4 The 1: 1 experimental group was allowed to add glucose 40% (w / v) and galactose 10% (w / v). The oil value culture is maintained at about 20 to 80% of the maximum dissolved oxygen, the concentration of remaining total reducing sugar (galactose + glucose) in the medium is maintained at 0.5 to 5% (w / v), and the concentration of glucose is 0.1 g / l or less. The feed rate of the liquid medium was adjusted to be. Samples were taken during batch and fed-batch cultures to analyze the absorbance at 600 nm, the measurement of carbon sources, and the amount of foreign protein (LK8) secreted (Table 1).

The determination of the carbon source was performed by measuring the residual amount of glucose and galactose in the medium. First, the measurement of glucose was performed using the glucose quantitative kit (Glucose-E, Young-Dong Pharm., Cat # BC103-E, Korea) using the enzyme reaction method as follows: Centrifuged samples were centrifuged at 12,000 rpm. Isolate and take the resulting supernatant. A coloring reagent was prepared by mixing a 100 ml dilution powder with 100 ml of PGO enzyme (peroxidase 100 U + glucose oxidase 500 U) included in the quantitative kit using the enzyme reaction method. After taking 3 ml separately from the coloring reagent prepared above, 20 μl of a glucose standard sample was added thereto. 20 µl was added to the sample to be measured in the same manner. It was reacted at 37 ° C. for 5 minutes and each absorbance was measured at a wavelength of 505 nm. The amount of glucose remaining in the medium was calculated by substituting the absorbance values of the glucose standard sample and the measured sample into the formula. The measurement of the total carbon source in the medium was performed using the DNS (3,5-dinitrosalicylic acid) method to determine the total reducing equivalent. Galactose and glucose are both reducing sugars and react equally to DNS equivalent reagents. 300 ml of 2 M sodium hydroxide, 0.25 g of 3,5-dinitrosalicylic acid, and 75 g of sodium potasium tartrate were added to 1 ml of the DNS reagent to be measured. After 100 μl of the sample was added, it was boiled for about 5 minutes and absorbance was measured at a wavelength of 550 nm to determine the total carbon source remaining in the medium. Measurement of the secreted foreign protein mass in the culture was analyzed by high performance liquid chromatography (HPLC) method as in Example 3 above.

Comparison of Production Yields per Unit Cell and Production Yields per Unit Induced by Transforming Yeast A with Different Galactose: Glucose Ratios Weight ratio of glucose to galactose in culture medium Total Galactose Consumption (g) LK8 secretion (mg / L) Cell growth (OD ₆₀₀ ) Yp / i ¹⁾ (mg / g) Yp / x ²⁾ (mg / L / OD) Control group (galactose only) 270 100 120 0.37 0.83 1: 4 200 114 100 0.57 1.14 1: 1 190 150 88 0.79 1.70 4: 1 78 44 77 0.56 0.57 ¹⁾ Yp / i: Inducer yield per unit inducer, foreign protein mass secreted per unit inducer (galactose). ²⁾ Yp / x: product yield per unit cell, secreted foreign protein mass per unit cell.

After batch culture, cell growth, carbon source consumption, and foreign protein secretion were compared between galactose alone and cultured by controlling the ratio of galactose and glucose. As shown in Table 1, when fed-batch culture using only galactose as a carbon source, foreign protein (LK8) was secreted extracellularly at a concentration of up to about 100 mg / l. It showed the highest cell growth (OD ₆₀₀ = 120) compared to the other experimental groups. When the milk was cultured by adjusting the concentration of glucose and galactose to 1: 4, the foreign protein showed a maximum secretion amount of 114 mg / L, and the amount of the foreign protein secreted per unit expression inducing substance (galactose) added was 40%. Increased. In the case of added glucose, all of them were exhausted within 24 hours of batch culture, and the glucose administered during fed-batch culture was immediately consumed to maintain the residual concentration below 0.1 g / L. When the milk was cultured at a ratio of 1: 1 adjusted to the same concentration of glucose and galactose, the secretion amount of LK8 was up to about 150 mg / L, which was the highest value compared with other experimental groups. Increases the production yield per unit cell (Yp / x, mg / L / OD ₆₀₀ ) and the yield per unit derivative (Yp / i, mg / g) to 104% and 114%, respectively. It was confirmed that the efficiency increased. When milk and cultured at a concentration of 4: 1 by reversing the concentration of glucose and galactose, the expression and secretion of foreign proteins were suppressed due to the high glucose ratio, resulting in a maximum secretion of only 44 mg / L.

Example 5 Inhibition of Galactose-Induced Promoter of Mixed Yeast B by Mixed-carbon Source Feeding and Comparison of Foreign Protein Secretion Yield

Except for using the transformed yeast B prepared in Example 2-2 as the strain to be cultured in the same manner as in Example 4 when using only the galactose as a carbon source when the transgenic yeast B is fed a milk value and galactose and Cell growth, carbon source consumption, and foreign protein secretion were compared when cultured by adjusting the glucose ratio (Table 2).

Comparison of Production Yields per Unit Cell and Sanic Acid Yields per Unit Induced by Transforming Yeast B with Different Values of Galactose: Glucose Weight ratio of glucose to galactose in culture medium Total Galactose Consumption (g) LK8 secretion (mg / L) Cell growth (OD ₆₀₀ ) Yp / i ¹⁾ (mg / g) Yp / x ²⁾ (mg / L / OD) Control group (galactose only) 800 250 62 0.31 4.03 1: 4 604 300 39 0.50 7.84 2: 3 287 350 47 1.22 7.44 1: 1 240 330 69 1.06 5.19 3: 2 180 140 50 0.53 2.8 ¹⁾ Yp / i: Inducer yield per unit inducer, exogenous protein secreted per unit inducer (galactose). ²⁾ Yp / x: product yield per unit cell, secreted foreign protein mass per unit cell.

As shown in Table 2, when galactose alone was incubated with a carbon source, foreign protein (LK8) was secreted extracellularly at a concentration of about 250 mg / L. When milk was cultured by adjusting the concentration of glucose and galactose to 1: 4, the foreign protein showed a maximum secretion amount of 300 mg / L. In the case of glucose, all of the glucose was exhausted within 24 hours of batch culture, and the glucose administered during fed-batch culture was immediately consumed. When glucose and galactose were incubated at a ratio of 2: 3, the maximum foreign protein secretion was obtained at 160 hours, and the absorbance showing cell growth was OD ₆₀₀ = 47. It was confirmed that both glucose and galactose were used as sugar scales. The maximum secretion of foreign protein was about 350 mg / L, which showed the shortest time to reach the maximum secretion compared to other experimental groups. At this time, the foreign protein secretion increased by 40% compared to the control, and the yield per unit cell (Yp / x, mg / L / OD ₆₀₀ ) and the yield per unit inducer (Yp / i, mg / g) were 293%, respectively. And increased to 85% was confirmed that dramatically increased foreign protein secretion efficiency. When the milk was incubated at a ratio of 1: 1 adjusted to the same concentration of glucose and galactose, the growth of cells was the most active. In addition, the secretion amount of LK8 was up to about 350 mg / L, which was quite high. However, when compared to the 2: 3 ratio experiment showing similar maximum secretion, it took nearly 380 hours to produce 350 mg / L in the 1: 1 experiment, which took about 2.5 times longer incubation time. Appeared. In the case of incubation at a ratio of 3: 2 by reversing the concentration of glucose and galactose, the growth of cells was relatively high and the consumption of sugar was also active. However, the expression and secretion of the foreign protein was suppressed due to the high glucose ratio, so the maximum secretion amount was only about 140 mg / L.

Example 6 In Yeast Strains A and B PDI1  Improved foreign protein secretion efficiency through gene overexpression

<Example 6-1> PDI1  Preparation of coexpression strains

<Example 6-1-1> PDI1  Preparation of Expression Vectors

Protein disulfide isomerase gene PDI1 coding for the enzyme in the same way as (protein disulfide isomerase, PDI) to so as to be expressed by induction galactose was inserted into an expression vector with the GAL10 promoter: First, 2u yeast origin of replication PESC-URA (Stratagene, USA) was digested with Mun I and Sna BI and treated with DNA polymerase (Klenow fragment, New England Biolabs, USA) to re-ligate 5.9 kb smooth terminal DNA fragments. To obtain the optimized vector pMK71. PCR was performed on the vector pMK71 using a chromosome derived from S. cerevisiae 2805 as a template with primers PDI1F: 5'-ATAAGAATGCGGCCGCCATACATCTATCCCGTTATGAAG-3 '(SEQ ID NO: 17) and PDI1R: 5'-GGACTAGTTTACAATTCATCGTGAATGGCATC-3' (SEQ ID NO: 18). This gave a 1.7 kb DNA fragment comprising the PDI1 gene set forth in SEQ ID NO: 9 having a Not I / Spe I cleavage site. Then, cutting the linearized pMK71 and DNA fragments of the 1.7 kb Not I and Spe I, respectively and ligating them to each other was prepared an expression vector of the PDI1 gene, it was named as pMPDI1 (Fig. 7). 7 shows protein disulfide isomerase by galactose when foreign proteins are expressed in yeast. A cleavage map of the pMPDI1 expression vector allowing PDI) to be co-expressed.

<Example 6-1-2> PDI1  Production of Co-expression LK8 Production Strains

The transgenic yeasts A and B were transformed with pMPDI1 prepared in Example 6-1-1, respectively. Transformation was performed using the alkaline cationic yeast transformation kit (Q-BIO gene, Canada) according to the manufacturer's instructions. The transformed yeast strains were named as S. cerevisiae 2805 / MδLK8 / PDI and S. cerevisiae BJ3501 / MδLK8 / PDI, respectively.

<Example 6-2> PDI1  Coexpression Effect and Comparison

In order to compare the effect of simultaneous expression of PDI1 gene on foreign protein secretion, transgenic yeast A and transformed yeast B and strains in which pMPDI1 was transduced into these strains ( S. cerevisiae 2805 / MδLK8 / PDI and S. cerevisiae BJ3501) / MδLK8 / PDI) were each taken out in storage at -70 ° C, and fed in the same manner as in Example 3 to induce the expression and secretion of foreign proteins (Table 3).

As shown in Table 3, the secreted LK8 of S. cerevisiae 2805 / MδLK8 is 100 mg / L and S. cerevisiae 2805 / MδLK8 / PDI1 of secretion in Saccharomyces to 130 mg / L My process three jiae Levy S. cerevisiae 2805 / MδLK8 / PDI1 is exhibited higher secretion. In addition, other strains of secretion of the S. cerevisiae BJ3501 / MδLK8 is 250 mg / L and S. cerevisiae BJ3501 / MδLK8 / secretion of the PDI1 has shown more is secreted from the PDI1 Again, co-expression strain with 430 mg / L higher. LK8 secretion of strains and non PDI1 co-expression strains from two different strains was higher in PDI1 gene co-expression strain.

Comparison of Extracellular Maximum Secretion of Foreign Proteins with Simultaneous Expression of Control and PDI1 Genes in Transgenic Yeasts A and B S. cerevisiae 2805 / MδLK8 S. cerevisiae BJ3501 / MδLK8 Control group ¹⁾ PDI1 co-expressing strain Control PDI1 co-expressing strain Maximum LK8 Secretion (mg / L) 100 130 250 430 ¹⁾ Control group: When PDI1 is not co-expressed

As claimed and demonstrated above, the present invention relates to a method for improving the secretion efficiency of recombinant foreign protein in a yeast expression system. The method for improving the secretion efficiency of the foreign protein of the present invention by appropriately controlling the level of galactose acting as an inducer of galactose-induced promoter in cells, recombination by overexpression generated in the yeast expression system based on the conventional galactose-induced promoter By reducing the insoluble precipitation of the foreign protein, ultimately to improve the secretion efficiency of the recombinant foreign protein, it can be usefully used to improve the productivity of the recombinant foreign protein in the yeast expression system, and to lower the production cost.

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Claims (21)

(Iii) transforming the host yeast with a recombinant foreign gene construct expressed under the influence of a galactose-induced promoter sequentially comprising a galactose-induced promoter, a secretion sequence and a gene encoding a foreign protein to produce a transformed yeast strain; And (Ii) culturing the transformed yeast strain under conditions in which the activity of the galactose-induced promoter is inhibited, The conditions under which the activity of the galactose-induced promoter of step (ii) is inhibited are either galactose by injecting glucose which induces catabolite repression of galactose and galactose-induced promoters into the medium or conditions in which the intracellular transport rate of galactose is reduced. A method for improving the secretion efficiency of foreign proteins, characterized in that the conditions for inhibiting promoter activity. The method of claim 1, wherein said step (iii) is performed by multiplexing said recombinant foreign gene construct into the chromosome of said host yeast or inserting it into a vector. The method of claim 1 wherein the galactose inducible promoters are characterized in that comprising the GAL7 promoter, GAL10 promoter described by SEQ ID NO: 3 or described in GAL1 promoter, SEQ ID NO: 2 described in SEQ ID NO: 1. According to claim 1, wherein the secretion sequence is the alpha (α) -secretion sequence of S. cerevisiae ( S. cerevisiae ) described in SEQ ID NO: 4, Saccharomyces cerevisiae described in SEQ ID NO: 5 K1 lethal toxin secretion (S. cerevisiae) sequence (killer toxin signal), SEQ ID NO: Inverted hydratase (invertase) secretion in Saccharomyces My process three Levy jiae (S. cerevisiae) as is described in SEQ ID NO: 6, SEQ ID NO: 7 is described in A lethal toxin secretion sequence of Klueveromyces lactis , a lethal toxin secretion sequence of Pichia acaciae described in SEQ ID NO: 8, a lethal toxin of Hanseniaspora uvarum Secretory sequence or lethal toxin secretion sequence of Pichia anomala . The method of claim 1, wherein the intracellular transport rate of galactose is reduced by the host yeast of step (iii) or the modified strain from which the galactose permease gene is artificially removed from the galactose inducing promoter. And transforming the recombinant foreign gene construct expressed under the influence, and culturing the transformed strain in a galactose-containing medium. The method of claim 1, wherein the intracellular transport rate of galactose is reduced by the host yeast of step (iii) or the modified strain from which the galactose permease gene is artificially removed from the galactose inducing promoter. And transforming the recombinant foreign gene construct expressed under influence, and culturing the transformed strain in a galactose and glucose-containing medium. The method of claim 1, wherein the medium is galactose and glucose The conditions for suppressing galactose promoter activity by injection are host yeasts of step (iii), which express normal yeast strain or galactose permease gene deficient strain or modified strain which has artificially removed galactose permease gene under the influence of galactose-induced promoter. Transformed with a recombinant foreign gene construct, wherein the transformed strain is a condition for culturing in a medium in which galactose and glucose are injected simultaneously. The method according to any one of claims 5 to 7, wherein the galactose permease gene defective strain is Saccharomyces cerevisiae BJ3501 (ATCC 208280). 8. The method of any one of claims 1, 6 and 7, wherein said galactose and glucose are injected by fed-batch culture. 10. The method of claim 9, wherein the galactose and glucose are injected in a weight ratio of galactose: glucose = 8: 1 to 1: 4. delete delete (Iii) transforming the host yeast with a recombinant foreign gene construct expressed under the influence of a galactose-induced promoter sequentially comprising a galactose-induced promoter, a secretion sequence and a gene encoding a foreign protein to produce a transformed yeast strain; And, (Ii) culturing the transformed yeast strain under conditions in which the activity of the galactose-induced promoter is inhibited by controlling the ratio of galactose and glucose, The galactose and glucose ratio is galactose: glucose = 8: 1 to 1: 4 (weight ratio) characterized in that the fed-batch culture method of recombinant yeast for improving the secretion of recombinant foreign protein, characterized in that. The method according to claim 13, wherein the galactose permease gene deletion strain is Saccharomyces cerevisiae BJ3501 (ATCC 208280). (Iii) Protein disulfide isomerases expressed under the influence of recombinant foreign gene constructs and galactose-induced promoters expressed under the influence of galactose-induced promoters that sequentially include genes encoding galactose-induced promoters, secretory sequences and foreign proteins. preparing a transformed yeast strain by transforming host yeast with a gene construct encoding protein disulfide isomerase; And (Ii) culturing the transformed yeast strain under conditions in which the activity of the galactose-induced promoter is inhibited, The conditions in which the activity of the galactose-induced promoter of step (ii) is inhibited are either galactose by injecting glucose which induces catabolite repression of galactose and galactose-induced promoters in a condition in which intracellular transport rate of galactose is reduced or medium. It is a condition which suppresses promoter activity, The method of improving secretion of a foreign protein. The method of claim 15, wherein the transformation of step (iii) is carried out sequentially or simultaneously. 16. The method according to claim 15, wherein the intracellular transport rate of galactose is reduced in the host yeast of step (iii), wherein the galactose permase gene deficient strain or the modified strain from which the galactose permease gene has been artificially removed from the galactose induction promoter. Transformed with a recombinant foreign gene construct expressed under the influence, and the transformed strain is characterized in that the culture conditions in a medium containing galactose containing medium or galactose: glucose = 8: 1 to 1: 4 weight ratio. . 16. The method according to claim 15, wherein the conditions for inhibiting galactose promoter activity by injecting galactose and glucose into the medium are normal yeast strains or galactose permease gene defective strains as host yeast of step (iii) or artificially galactose permease gene. The transformed strain was removed and transformed with a recombinant foreign gene construct expressed under the influence of a galactose-induced promoter, characterized in that the transformed strain is cultured in a medium in which galactose and glucose are injected at the same time. The method according to claim 17 or 18, wherein the galactose permease gene deletion strain is Saccharomyces cerevisiae BJ3501 (ATCC 208280). The method of claim 15, wherein the gene encoding the protein disulfide isomerase (hereinafter referred to as 'PDI') is a PDI1 , SEQ ID NO of S. cerevisiae (SEQ ID NO: 9) PDI of Conus textile described in 10, PDI of Caenorhabditis elegans described in SEQ ID NO: 11, human pancreatic PDI gene described in SEQ ID NO: 12, Aspergillus, a fungus described in SEQ ID NO: 13 PDI of Aspergillus oryzae , PDI of Candida boidinii described in SEQ ID NO: 14 or PDI of Humicola insolens described in SEQ ID NO: 15. 16. The method of claim 15, wherein the gene construct encoding a protein disulfide isomerase is pMPDI having a cleavage map as shown in FIG.

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