KR20060110546A - Methods for enhancing a secretion efficiency of recombinant foreign protein in yeast expression system - Google Patents

Abstract

Methods for enhancing the secretion efficiency of a recombinant foreign protein in the yeast expression system are provided to reduce precipitation of the recombinant foreign protein caused by its overexpression by controlling the level of galactose as an inducing material of a galactose induction promoter, and reduce the production costs of the recombinant foreign protein. The method for enhancing the secretion efficiency of a recombinant foreign protein in the yeast expression system comprises the steps of: (i) transforming host yeast with a recombinant foreign gene construct sequentially containing galactose induction promoter, secretion sequence and foreign protein gene whose expression is affected by the galactose induction promoter to produce the transformed yeast; and (ii) culturing the transformed yeast under the condition to control the activity of galactose induction promoter, wherein the galactose induction promoter is GAL1 promoter of SEQ ID NO:1, GAL10 promoter of SEQ ID NO:2 or GAL7 promoter of SEQ ID NO:3; the secretion sequence is alpha-secretion sequence of S. cerevisiae(SEQ ID NO:4), K1 killer toxin signal of S. cerevisiae(SEQ ID NO:5), invertase secretion sequence of S. cerevisiae(SEQ ID NO:6), killer toxin signal of Klueveromyces lactis(SEQ ID NO:7), killer toxin signal of Pichia acaciae(SEQ ID NO:8), killer toxin signal of Hanseniaspora uvarum or killer toxin signal of Pichia anomala; and the condition to control the activity of galactose induction promoter is the condition to reduce the transfer speed of galactose in a cell.

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, overexpression of proteins may result in inactivation of the inactivated state in the cell. Many factors are provided. 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 (i) 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. Glucose is not particularly limited thereto, but is preferably injected 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 according to time when galactose was supplied to a single carbon source in two host yeasts having the same genetic background except for the deletion of the GAL2 gene. As shown in Figure 3, it can be seen that in the host yeast B lacking the GAL2 gene, the maximum consumption rate and cell growth rate of galactose per unit time are reduced compared to the host yeast B in which the GAL2 gene is normally operating.

<Example 2> Preparation of transgenic yeasts secreting foreign proteins using host yeasts A and B as hosts

<Example 2-1> Exogenous protein secretion-type conversion yeast using host yeast A S. cerevisiae  Manufacture 2805 / MδLK8

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 galacose 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. In the case of milk culture at a ratio of 1: 1 adjusted to the same concentration of galactose and glucose, LK8 secretion 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 the lactose and glucose concentrations were reversed and fed in the milk at a concentration of 4: 1, the expression and secretion of the foreign protein were suppressed due to the high glucose ratio, and the maximum secretion was only about 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.

<110> Magam Biotechnology Institute <120> Methods for enhancing a secretion efficiency of recombinant          foreign protein in yeast expression system <130> 4p-10-03 <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 453 <212> DNA <213> Saccharomyces cerevisiae <220> <221> promoter (222) (1) .. (453) <223> GAL1 promoter <400> 1 agtacggatt agaagccgcc gagcgggtga cagccctccg aaggaagact ctcctccgtg 60 cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc cgcactgctc 120 cgaacaataa agattctaca atactagctt ttatggttat gaagaggaaa aattggcagt 180 aacctggccc cacaaacctt caaatgaacg aatcaaatta acaaccatag gatgataatg 240 cgattagttt tttagcctta tttctggggt aattaatcag cgaagcgatg atttttgatc 300 tattaacaga tatataaatg caaaaactgc ataaccactt taactaatac tttcaacatt 360 ttcggtttgt attacttctt attcaaatgt aataaaagta tcaacaaaaa attgttaata 420 tacctctata ctttaacgtc aaggagaaaa aac 453 <210> 2 <211> 507 <212> DNA <213> Saccharomyces cerevisiae <220> <221> promoter (222) (1) .. (507) <223> GAL10 promoter <400> 2 tcaaaaatca tcgcttcgct gattaattac cccagaaata aggctaaaaa actaatcgca 60 ttatcatcct atggttgtta atttgattcg ttcatttgaa ggtttgtggg gccaggttac 120 tgccaatttt tcctcttcat aaccataaaa gctagtattg tagaatcttt attgttcgga 180 gcagtgcggc gcgaggcaca tctgcgtttc aggaacgcga ccggtgaaga cgaggacgca 240 cggaggagag tcttccttcg gagggctgtc acccgctcgg cggcttctaa tccgtacttc 300 aatatagcaa tgagcagtta agcgtattac tgaaagttcc aaagagaagg tttttttagg 360 ctaagataat ggggctcttt acatttccac aacatataag taagattaga tatggatagt 420 tatatggata tgtatatggt ggtaatgcca tgtaatatga ttattaaact tctttgcgtc 480 catccaaaaa aaaagtaaga atttttg 507 <210> 3 <211> 625 <212> DNA <213> Saccharomyces cerevisiae <220> <221> promoter (222) (1) .. (625) <223> GAL7 promoter <400> 3 ggagttcagt gataaaagtg tcacagcgaa tttcctcata tgtagggacc gaattgttta 60 caagttctct gtaccaccat ggagacatca aagattgaaa atctatggaa agatatggac 120 ggtagcaaca agaatatagc acgagccgcg gatttatttc gttacttttg atatcactca 180 caactattgc gaagcgcttc agtgaaaaaa tcataaggaa aagttgtaaa tattattggt 240 actattcgtt tggtaaagta gagggggtaa tttttcccct ttattttgtt catacattct 300 taaattgctt tgcctctcct tttggaaagc tatacttcgg agcactgttg agcgaaggct 360 cattagatat attttctgtc attttcctta acccaaaaat aagggagagg gtccaaaaag 420 cgctcggaca actgttgacc gtgatccgaa ggactggcta tacagtgttc acaaaatagc 480 caagctgaaa ataatgtgta gcctttagct atgttcagtt agtttggcta gcaaagatat 540 aaaagcaggt cggaaatatt tatgggcatt attatgcaga gcatcaacat gataaaaaaa 600 acagttgaat attccctcaa aaatg 625 <210> 4 <211> 85 <212> PRT <213> Saccharomyces cerevisiae <400> 4 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser   1 5 10 15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Thr Glu Asp Glu Thr Ala Gln              20 25 30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe          35 40 45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu      50 55 60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val  65 70 75 80 Ser Leu Glu Lys Arg                  85 <210> 5 <211> 26 <212> PRT <213> Saccharomyces cerevisiae killer virus M1 <400> 5 Met Thr Lys Pro Thr Gln Val Leu Val Arg Ser Val Ser Ile Leu Phe   1 5 10 15 Phe Ile Thr Leu Leu His Leu Val Val Ala              20 25 <210> 6 <211> 19 <212> PRT <213> Saccharomyces cerevisiae <400> 6 Met Leu Leu Gln Ala Phe Leu Phe Leu Leu Ala Gly Phe Ala Ala Lys   1 5 10 15 Ile ser ala            <210> 7 <211> 17 <212> PRT <213> Kluyveromyces lactis <400> 7 Met Asn Ile Phe Tyr Ile Phe Leu Phe Leu Leu Ser Phe Val Gln Gly   1 5 10 15 Leu     <210> 8 <211> 48 <212> PRT <213> Pichia acaciae <400> 8 Met Leu Ile Ile Val Leu Leu Phe Leu Ala Thr Leu Ala Asn Ser Leu   1 5 10 15 Asp Cys Ser Gly Asp Val Phe Phe Gly Tyr Thr Arg Gly Asp Lys Thr              20 25 30 Asp Val His Lys Ser Gln Ala Leu Thr Ala Val Lys Asn Ile Lys Arg          35 40 45 <210> 9 <211> 1569 <212> DNA <213> Saccharomyces cerevisiae <300> Tachikawa, H. et al. <302> Molecular structure of a yeast gene, PDI1, encoding protein          disulfide isomerase that is essential for cell growth J. Biochem. <304> 110 <305> 2 <306> 306-313 <400> 9 atgaagtttt ctgctggtgc cgtcctgtca tggtcctccc tgctgctcgc ctcctctgtt 60 ttcgcccaac aagaggctgt ggcccctgaa gactccgctg tcgttaagtt ggccaccgac 120 tccttcaatg agtacattca gtcgcacgac ttggtgcttg cggagttttt tgctccatgg 180 tgtggccact gtaagaacat ggctcctgaa tacgttaaag ccgccgagac tttagttgag 240 aaaaacatta ccttggccca gatcgactgt actgaaaacc aggatctgtg tatggaacac 300 aacattccag ggttcccaag cttgaagatt ttcaaaaaca gcgatgttaa caactcgatc 360 gattacgagg gacctagaac tgccgaggcc attgtccaat tcatgatcaa gcaaagccaa 420 ccggctgtcg ccgttgttgc tgatctacca gcttaccttg ctaacgagac ttttgtcact 480 ccagttatcg tccaatccgg taagattgac gccgacttca acgccacctt ttactccatg 540 gccaacaaac acttcaacga ctacgacttt gtctccgctg aaaacgcaga cgatgatttc 600 aagctttcta tttacttgcc ctccgccatg gacgagcctg tagtatacaa cggtaagaaa 660 gccgatatcg ctgacgctga tgtttttgaa aaatggttgc aagtggaagc cttgccctac 720 tttggtgaaa tcgacggttc cgttttcgcc caatacgtcg aaagcggttt gcctttgggt 780 tacttattct acaatgacga ggaagaattg gaagaataca agcctctctt taccgagttg 840 gccaaaaaga acagaggtct aatgaacttt gttagcatcg atgccagaaa attcggcaga 900 cacgccggca acttgaacat gaaggaacaa ttccctctat ttgccatcca cgacatgact 960 gaagacttga agtacggttt gcctcaactc tctgaagagg cgtttgacga attgagcgac 1020 aagatcgtgt tggagtctaa ggctattgaa tctttggtta aggacttctt gaaaggtgat 1080 gcctccccaa tcgtgaagtc ccaagagatc ttcgagaacc aagattcctc tgtcttccaa 1140 ttggtcggta agaaccatga cgaaatcgtc aacgacccaa agaaggacgt tcttgttttg 1200 tactatgccc catggtgtgg tcactgtaag agattggccc caacttacca agaactagct 1260 gatacctacg ccaacgccac atccgacgtt ttgattgcta aactagacca cactgaaaac 1320 gatgtcagag gcgtcgtaat tgaaggttac ccaacaatcg tcttataccc aggtggtaag 1380 aagtccgaat ctgttgtgta ccaaggttca agatccttgg actctttatt cgacttcatc 1440 aaggaaaacg gtcacttcga cgtcgacggt aaggccttgt acgaagaagc ccaggaaaaa 1500 gctgctgagg aagccgatgc tgacgctgaa ttggctgacg aagaagatgc cattcacgat 1560 gaattgtaa 1569 <210> 10 <211> 1601 <212> DNA <213> Conus textile <400> 10 gaattcgccc ttactaggat ccgcatcatc atgaagtttt catcttgttt agttttaact 60 cttctggttt ttgtatctgc cgaagatgtc gaacaggagg aaaatgtcca cgttttgacg 120 aagaaaaatt ttgactcctt cataactgat aatgagttcg tgcttgtgga attttatgct 180 ccctggtgtg gccattgcaa ggcattggca ccagaatatg ccaaagctgc aacaactttg 240 gaaaacgaga agtcgaacat caagttggcc aaagtggatg ctactgtgga gggggatttg 300 gcctccaaat ttgatgttcg tggataccca acaatcaagt tcttccgtaa agagaagcct 360 gatggtccag cagactacag tggtggtcgc caagctaaag atattgttga ctggctgaag 420 aagaagacag gaccaccagc caaggaactg aaggagaaag atgaagtcaa ggcttttgtg 480 gaaaaagatg aagttgttgt cattggtttc ttcaaggatc aagaatccac aggtgctttg 540 gccttcaaaa aggcagctgc cggcattgat gacattccat ttgccatcac ttcagaagat 600 catgttttca aggagtacaa gatggacaaa gatggcattg tactgctgaa gaagtttgat 660 gagggccgta atgacttcga ggggaatttg gaggaggagg aggccatcgt caagcacgtc 720 agggaaaacc aactgccact ggttgtagaa ttcactcaag agtctgccca gaagatcttt 780 ggaggtgagg tgaagaacca cattctgctg ttcctgaaga aggaaggtgg agaagacaca 840 attgaaaagt tcagaagtgc agctgaggat ttcaaaggaa aggtcctgtt tatctacttg 900 gacactgaca atgaggagaa tggacgcatc acagagttct ttggcttgaa ggatgatgaa 960 atcccagctg tgcgtctgat ccagctggca gaggacatgt caaagtacaa gcctgagtcc 1020 tcggatttgg aaactgccac catcaagaaa tttgtccagg atttcctgga tgggaaactg 1080 aagccccatc tgatgtctga ggatgtgcct ggtgactggg atgccaagcc tgtgaaggtc 1140 ctggtgggca agaacttcaa ggaagtggcg atggacaaat caaaggctgt ctttgtggag 1200 ttctatgctc cctggtgtgg acactgcaag cagctggccc ctatctggga tgagctgggt 1260 gaaaagtaca aggacagcaa ggacattgtt gttgccaaga tggatgccac tgccaatgag 1320 attgaagagg tcaaagtgca gagcttcccc accctcaagt acttccccaa ggacagcgag 1380 gaggctgtgg actacaatgg cgagagaacc ttggatgctt tcgttaaatt cctcgagagc 1440 ggtggcacgg aaggtgctgg agtgcaagag gatgaggaag aggaagagga agatgaggag 1500 ggtgatgatg aagatctgcc aagagatgaa ctgtagctgt catcggcatc tagactcgaa 1560 gggcgaattc cagcacactg gcggccgtta ctagtggatc c 1601 <210> 11 <211> 1458 <212> DNA <213> Caenorhabditis elegans <300> Page, A. P. <302> Cyclophilin and protein disulfide isomerase genes are          co-transcribed in a functionally related manner in Caenorhabditis          elegans <303> DNA Cell Biology <304> 16 <305> 11 <306> 1335-1343 <400> 11 atgtcactgt cagtgtcctt tatcttcctc ctggtcgcat caatcggagc agttgttgct 60 gacagcgaaa acgtgcttgt tctcactgaa agcaatttcg aagaaactat caatggaaat 120 gagtttgttc tagtcaaatt ctatgctcca tggtgtgtac attgcaagtc tcttgctcca 180 aagtacgacg aagccgccga tttgctgaag gaggaaggat ccgatatcaa gctcgccaag 240 gttgacgcca ccgaaaacca agctcttgca tccaagttcg aggtccgtgg atatccaact 300 atcctctact tcaagagtgg aaagccaacg aaatacaccg gaggacgcgc caccgctcaa 360 attgtcgatt gggtcaagaa gaagagtgga ccaactgtta ccactgttga gtcagttgag 420 caactcgagg aattgaaggg aaagaccaga gttgttgttc ttggatactt caaggacgcg 480 aaatctgatg ctgctaccat ctacaacgaa gttgctgatt ctgtcgatga cgcattcttc 540 gccgttgccg gttctgctga ggttgctgct gccgcatctt tgaatgaaga tggagtcgct 600 cttatccgca ctgatggaga cgacagcgag actagcacaa ttgctgaagc tgaaatcacc 660 aacaccatcg ctcttaagca atggctccac gcttacaagc tctctgccgt gaccgagttc 720 acccacgaat ctgctcaaga aatcgtcgga ggagacctca aaaagttcca ctttttgatc 780 atccgcaagt cagactcttc tttcgatgag actattgcca agttcactga ggtcgctaag 840 aagttccgtg ctaagatcgt ctttgttctt ctcgacgttg atgttgaaga gaacgcaaga 900 attctcgagt tcctcggagt cgatgccaag aacaccccag ccaacagaat tgtcagcctt 960 gccgatcaag ttgagaagtt caagccacaa gaaggagaag atttcgaagc tttcaccaac 1020 tcatatttgg aaggaaagtc tgctcaagac ttgaaggctc aagatcttcc agaagattgg 1080 aatgcactcc cagttaaggt tctcgtcgcc tccaacttca acgaaattgc ccttgatgaa 1140 accaagactg tcttcgtcaa attctacgcc ccatggtgcg gacattgcaa gcaactggtt 1200 ccagtttggg atgagcttgc tgagaagtac gagtccaacc caaatgttgt cattgcaaag 1260 cttgatgcta ctctcaacga gcttgccgac gtcaaggtca actctttccc aaccctgaag 1320 ctgtggccag ccggatcttc taccccagtc gactatgatg gagacagaaa cctcgagaag 1380 ttcgaagaat ttgtcaacaa gtatgctgga tctgcttccg aatctgaaac cgcatctcag 1440 gatcacgaag agctttaa 1458 <210> 12 <211> 1536 <212> DNA <213> Homo sapiens <300> <301> Desilva, M. G. et al. <302> Characterization and chromosomal localization of a new protein          disulfide isomerase, PDIp, highly expressed in human pancreas <303> DNA Cell Biology <304> 15 <305> 1 <306> 9-16 <400> 12 atggcttcgt gcccatgggg tcaggaacag ggagcgagga gcccctcgga ggagcctcca 60 gaggaggaaa tccccaagga ggatgggatc ttggtgctga gccgccacac cctgggcctg 120 gccctgcggg agcaccctgc cctgctggtg gaattctatg ccccgtggtg tgggcactgc 180 caggccctgg cccccgagta cagcaaggca gctgccgtgc tcgcggccga gtcaatggtg 240 gtcacgctgg ccaaggtgga tgggcccgcg cagcgcgagc tggctgagga gtttggtgtg 300 acggagtacc ctacgctcaa gttcttccgc aatgggaacc gcacgcaccc cgaggagtac 360 acaggaccac gggacgctga gggcattgcc gagtggctgc gacggcgggt ggggcccagt 420 gccatgcggc tggaggatga ggcggccgcc caggcgctga tcggtggccg ggacctagtg 480 gtcattggct tcttccagga cctgcaggac gaggacgtgg ccaccttctt ggccttggcc 540 caggacgccc tggacatgac ctttggcctc acagaccggc cgcggctctt tcagcagttt 600 ggcctcacca aggacactgt ggttctcttc aagaagtttg atgaggggcg ggcagacttc 660 cccgtggacg aggagcttgg cctggacctg ggggatctgt cgcgcttcct ggtcacacac 720 agcatgcgcc tggtcacgga gttcaacagc cagacgtctg ccaagatctt cgcggccagg 780 atcctcaacc acctgctgct gtttgtcaac cagacgctgg ctgcgcaccg ggagctccta 840 gcgggctttg gggaggcagc tccccgcttc cgggggcagg tgctgttcgt ggtggtggac 900 gtggcggccg acaatgagca cgtgctgcag tactttggac tcaaggctga ggcagccccc 960 actctgcgct tggtcaacct tgaaaccact aagaagtatg cgcctgtgga tgggggccct 1020 gtcaccgcag cgtccatcac tgctttctgc catgcagtcc tcaacggcca agtcaagccc 1080 tatctcctga gccaggagat accccctgat tgggatcagc ggccagttaa gaccctcgtg 1140 ggcaagaatt ttgagcaggt ggcttttgac gaaaccaaga atgtgtttgt caagttctat 1200 gccccgtggt gcacccactg caaggagatg gcccctgcct gggaggcatt ggctgagaag 1260 taccaagacc acgaggacat catcattgct gagctggatg ccacggccaa cgagctggat 1320 gccttcgctg tgcacggctt ccctactctc aagtacttcc cagcagggcc aggtcggaag 1380 gtgattgaat acaaaagcac cagggacctg gagactttct ccaagttcct ggacaacggg 1440 ggcgtgctgc ccacggagga gtccccggag gagccagcag ccccgttccc ggagccaccg 1500 gccaactcca ctatggggtc caaggaggaa ctgtag 1536 <210> 13 <211> 1547 <212> DNA <213> Aspergillus oryzae <400> 13 atgcggactt tcgcaccttg gatcttgagc cttctagggg cttctgctgt agcttctgct 60 gccgatgcga ctgccgaagc tccctccgat gtggtctcgc tcaccgggga cacattcgaa 120 actttcgtca aggagcatga cctagttttg gccgagtttt ttgctccctg gtgtggccat 180 tgcaaggctc tcgctccgaa atacgagcag gccgccactg agttaaagga aaagaacatt 240 ccgctggtca aggttgattg caccgaggaa gaggctcttt gtagggacca aggtgttgaa 300 ggttacccca cgctgaagat tttccgtggc cttgacgctg ttaagcctta tcagggagct 360 cgtcagaccg aggcgattgt ttcatacatg gtcaagcagt cactacctgc tgtgtcccct 420 gtcaccccag aaaacctcga agagatcaag actatggaca agattgtcgt tattggttat 480 atcgcgtctg acgaccagac tgccaatgat atattcacca cttttgccga gtcacagaga 540 gacaactacc tcttcgccgc cacaagtgat gcatcgatcg ctaaggcaga aggtgttaag 600 caaccttcga ttgttctcta taaagacttc gatgaaaaga aagctactta tgatggagag 660 attgaacagg atgccctcct cagttgggtc aagactgcca gtaccccctt ggtgggcgag 720 ctgggcccag agacttactc cggatatata acggctggca ttccactggc gtacattttc 780 gccgaaacca aagaagagcg tgagcagttc accgaggagt tcaagttcat cgccgagaaa 840 cacaagggtt ccatcaatat tgtcaccatt gacgccaagt tgtacggcgc tcatgcaggc 900 aatctcaacc ttgacccctc caagttccct gcattcgcta ttcaagaccc tgaaaagaac 960 gccaagtatc cttatgacca gtcgaaggaa gtcaaggcca aggatatcgg taaattcatc 1020 caagacgttc ttgatgataa agtagagcca agcattaagt ctgaggctat tcctgagact 1080 caggaaggtc ctgttactgt tgttgtcgcg cattcctata aggatctcgt ccttgacaac 1140 gagaaggacg tccttctcga attttatgcg ccatggtgcg gacactgcaa ggcccttgcc 1200 ccgaagtacg aggaacttgc aagcctttac aaggatattc ctgaagttac catcgccaaa 1260 attgacgcaa cggccaacga tgtccccgac tccattacag gatttcctac tattaagctc 1320 ttcgctgccg gcgccaagga ctccccagtt gaatatgaag gctctcgcac ggtggaggac 1380 ctcgccaact tcgtcaagga gaatggcaag cacaaggtcg atgctcttga agttgatccg 1440 aagaaagaac aggagagtgg cgatgccacc gagactcggg ccgcctctga cgagaccgaa 1500 actcctgctg ctactagcga tgacaagtct gagcatgatg aattgta 1547 <210> 14 <211> 1593 <212> DNA Candida boidinii <400> 14 atgaagttaa ctaatttcaa agttattgcc acaattcttg cttgtttaac agttgttaga 60 gctgatgatg gtggtgccat tgcatctcca gattccgctg ttgttaaatt aactgctgat 120 tcattcgaat cattcatgaa agaaaatcca ttagtcttag ctgaattttt tgctccttgg 180 tgtggtcatt gtaaaagatt gggtcctgaa tttcaagttg ctgctgataa attagttgaa 240 aaagatatta gattagctca aattgattgt accgaagaaa aagatttatg ttcttcttat 300 ggtattaaag gttacccaac tttaaaagtc tttagaggtt acgaaaatga accttctgat 360 tatgctggtc aaagaacttc agattcaatc atttcttata tggttaaaca atcaacccca 420 cctgtctcca tcgttgatga tctctcagat atcgaagata caattaaaga atcaaatgat 480 cctgtcttta ttcaagtctt accaaaaggt tctaaatctg ttgaagccgg taactcaact 540 ttctttgaaa tcgctaatgg tttaagagat aactactctt ttatttcaac aacaagtact 600 gaattctctt caaaatactt gaaaggtatt aaaaaatcag atactccatc ttatattctc 660 tttagaccaa atgaagaatt gtctgatgct tcaatctata aatttgatga aattgatgat 720 actcatttaa tcgaattctt aaacgttgaa tcaaaacctt tattcggtga aatggatggt 780 tcttctttcc aatcttatat ggaaatgaaa ttaccagttg cttattattt ctataatgaa 840 atctctgaaa aagatgccgt ctctgatgcc atcagtaaat tagctaaaac tcatagaggt 900 aaagttaatt tcgttggttt agacgcttct aaatatggtt tacacgctaa gaatattaac 960 atgaaggaag aattccctct tttcgctatt cacgatttag caactgaatt aaaatacggt 1020 atctcccaag ataaaccatt agataataaa ttaattccaa aattcgttga agatttcgtt 1080 gctggtaaat tagaagcaat cattaaatca gaaccaatcc cagaaactca agattctcca 1140 gtttaccatt tagtcggtaa agaacatgat aaaattatta cctctgataa agatgtctta 1200 gttaaatatt acgctccatg gtgtggtcac tgtaaaaaat tagctccagt ctttgaagaa 1260 ttagctgctg tttatgaatc agttgctcca ggtaaagtct tattagctga tttagatcat 1320 actgaaaatg atgtcaccgg tgttcacatt gaaggttacc caactatcgt cttataccca 1380 gccgatggtt cagaaccagt tgtttacgaa ggtaacagat cttttgaatc tttctccgat 1440 ttcattaaag aaaaaggttc atcaggtgtt gatgctaatg cattaaaaga accttaccca 1500 gaagaaggta ctgaaggtgc tccagttgat ccagaatcag ttggtgatgc tgaaaaagaa 1560 gatgattctg ctgctgatgt tcgtgatgaa tta 1593 <210> 15 <211> 1518 <212> DNA <213> Humicola insolens <400> 15 atgcataagg cccagaagtt cgcgctcggc ctgcttgccg cggcggcagt tgccacagct 60 tcggatgttg tccagctgaa gaaggacacc ttcgacgact tcatcaagac gaatgacctt 120 gttctcgccg aattcttcgc gccgtggtgc ggtcactgca aggctctcgc ccccgagtac 180 gaggaggctg cgaccacact gaaggagaag aacatcaagc tcgccaaggt ggactgcaca 240 gaggagacgg acctctgcca acaacatggt gttgagggct acccgactct caaggtcttc 300 cgcggccttg acaacgtctc cccctacaag ggccagcgca aggctgctgc tatcacctcg 360 tacatgatca agcagtctct gcccgccgtg tccgaggtca cgaaggacaa cctggaggag 420 ttcaagaagg ccgacaaggc cgtccttgtc gcctatgtgg atgcttccga caaggcgtcc 480 agtgaggttt tcacccaggt cgccgagaag ctgcgcgaca actacccgtt cggctccagc 540 agcgatgctg cgctggccga ggctgagggc gtcaaggctc ccgctatcgt cctttacaag 600 gactttgatg agggcaaggc ggtcttctcc gagaagttcg aggtggaggc gatcgagaag 660 ttcgccaaga cgggcgccac cccgctcatt ggcgagattg gccccgaaac ctactccgac 720 tacatgtcgg ccggcatccc tctggcctac attttcgccg aaacggccga ggagcggaag 780 gagctcagcg acaagctcaa gccgatcgcc gaggctcagc gcggcgtcat taactttggt 840 actattgacg ccaaggcttt tggtgcccac gccggcaacc tgaacctgaa gaccgacaag 900 ttccccgcct tcgccatcca ggaggtcgcc aagaaccaga agttcccctt cgatcaggag 960 aaggagatca ccttcgaggc gatcaaggct ttcgtcgacg actttgtcgc cggtaagatc 1020 gagcccagca tcaagtcgga gccgatccct gagaagcagg agggccccgt caccgtcgtc 1080 gttgccaaga actacaatga gatcgtcctg gacgacacca aggatgtgct gattgagttc 1140 tacgccccgt ggtgcggcca ctgcaaggcc ctggctccca agtacgagga gctcggcgcc 1200 ctgtatgcca agagcgagtt caaggaccgg gtcgtcatcg ccaaggttga tgccacggcc 1260 aacgacgttc ccgatgagat ccagggattc cccaccatca agctgtaccc ggccggtgcc 1320 aagggtcagc ccgtcaccta ctctggctcg cgcactgtcg aggacctcat caagttcatc 1380 gccgagaacg gcaagtacaa ggccgccatc tcggaggatg ccgaggagac gtcgtccgca 1440 accgagacga ccaccgagac ggccaccaag tcggaggagg ctgccaagga gacggcgacg 1500 gagcacgacg agctctag 1518 <210> 16 <211> 258 <212> DNA <213> Homo sapiens <220> <221> gene (222) (1) .. (258) <223> LK8 cDNA <400> 16 gaacaggact gcatgtttgg gaatgggaaa ggataccggg gcaagaaggc aaccactgtt 60 actgggacgc catgccagga atgggctgcc caggagcccc atagacacag cacgttcatt 120 ccagggacaa ataaatgggc aggtctggaa aaaaattact gccgtaaccc tgatggtgac 180 atcaatggtc cctggtgcta cacaatgaat ccaagaaaac tttttgacta ctgtgatatc 240 cctctctgtg catcctct 258 <210> 17 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Foward Primer for S. cerevisiae PDI1 (PDI1F) <400> 17 ataagaatgc ggccgccata catctatccc gttatgaag 39 <210> 18 <211> 32 <212> DNA <213> Artificial Sequence <220> Reverse primer for S. cerevisiae PDI1 (PDI1R) <400> 18 ggactagttt acaattcatc gtgaatggca tc 32  

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 controlled. 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 said secretory sequence is the α- secretory sequence, SEQ ID NO: K1 lethal toxin secretory sequence (killer toxin signal) of S. cerevisiae described in 5, SEQ ID NO: 6 of S. cerevisiae is described in SEQ ID NO: 4 Invertase secretion sequence of S. cerevisiae described, lethal toxin secretion sequence of Klueveromyces lactis as shown in SEQ ID NO: 7, lethal toxin secretion sequence of Pichia acaciae as shown in SEQ ID NO: 8, or lethal toxin secretion sequence of Hanseniaspora uvarum , or Pichia anomala method of lethal toxin secretion sequence. The method of claim 1, wherein the condition in which the activity of the galactose inducing promoter is regulated in step (ii) is a condition in which the intracellular transport rate of galactose is reduced. 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 6, wherein the galactose permease gene deletion strain is Saccharomyces cerevisiae BJ3501 (ATCC 208280). The method according to claim 1, wherein the condition in which the activity of the galactose-induced promoter is regulated in step (ii) is a condition of simultaneously injecting glucose into the medium to induce cataolite repression of galactose and galactose-induced promoter. How to. 9. The method of claim 8, wherein the galactose and glucose are injected by fed-batch culture. 10. The method of claim 9, wherein the galactose and glucose are injected at a ratio of 8: 1 to 1: 4. 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 to be expressed under influence, and injecting the transformed strain into a fed-batch galactose and glucose. 12. The method of claim 11, wherein galactose and glucose are injected at a ratio of 8: 1 to 1: 4. (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) a fed-batch culture method of recombinant yeast for improving the secretion of recombinant foreign protein, comprising culturing the transformed yeast strain by controlling the ratio of galactose and glucose. The culture method according to claim 13, wherein the host yeast of step (iii) is a galactose permease gene deficient strain or a modified strain from which the galactose permease gene has been artificially removed. (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 controlled. The method of claim 15, wherein the transformation of step (iii) is carried out sequentially or simultaneously. 16. The galactose-inducing promoter of claim 15, wherein the intracellular transport rate of galactose is reduced by a host yeast of the stage of the galactose deficient strain or a modified strain from which the galactose permease gene has been artificially removed. And transforming the recombinant foreign gene construct expressed under the influence of the condition, characterized in that the transformed strain is cultured in a galactose-containing medium. 18. The method of claim 17, wherein the galactose permease gene deletion strain is Saccharomyces cerevisiae BJ3501 (ATCC 208280). 16. The method according to claim 15, wherein the condition in which the activity of the galactose inducing promoter is regulated in step (ii) is a condition of simultaneously injecting glucose into the medium to induce catabolite repression of galactose and galactose inducing promoter. How to. The method of claim 15, wherein the protein disulfide isomerase (protein disulfide isomerase) coding genes are described in PDI, SEQ ID NO: 11 of the Conus textile described as PDI1, SEQ ID NO: 10 of S. cerevisiae are described in SEQ ID NO: 9, C. elegans is a PDI, SEQ ID NO: 12 is described as a human pancreatic PDI gene, PDI or SEQ ID NO: 15 of Candida boidinii which is described in the fungus Aspergillus oryzae of PDI, SEQ ID NO: 14 which is described in SEQ ID NO: 13 described in Humicola insolens The method characterized in that the PDI . 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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