KR100288889B1 - Method for preparing human serum albumin using transformed yeast Hannula polymorpha - Google Patents

Abstract

The present invention assembles an expression vector using an albumin cDNA comprising a secretion signal of albumin itself and a MOX promoter derived from yeast Hanshenula polymorpha DL-1, and the expression vector is assembled by introducing it into the yeast Hanshenula polymorpha. The present invention relates to a method for producing human serum albumin, an expression vector, and a transformed yeast, characterized by expressing human serum albumin with high efficiency by selecting and fermenting cultured transformed yeast.

Description

Method for producing human serum albumin using transformed yeast Hanshenula polymorpha

The present invention relates to an albumin expression vector for efficiently mass production of recombinant human serum albumin in a yeast Hansenula polymorpha and a method for producing human serum albumin using a transformed strain containing the same. More specifically, the expression vector is assembled using an albumin cDNA comprising a secretion signal of the MOX promoter and albumin itself, and then the expression vector is introduced into a yeast Hanshenula polymorpha, and the assembled expression vector is integrated into a chromosome. The present invention relates to a method for producing albumin by screening a sieve, and selecting a transformed strain from which the albumin is produced with high efficiency.

Human serum albumin (HSA) is a 66.6 kDa molecular weight protein consisting of 585 amino acids. Albumin, which accounts for about 60% of the plasma protein content, maintains the osmotic pressure of the plasma, as well as transporting fatty acids, bile pigments, amino acids, steroid hormones, and metal ions (Peters, T. The plasma proteins, ed. FW) Putnam, p. 133 (1975), Academic Press Inc. Albumin is used as an alternative to plasma expansion or as a blood replenishment when bleeding, and medically, the demand is increasing. Until now, human serum albumin is mainly purified and purified from blood, but stability problems caused by contamination of pathogens and viruses have recently emerged, and mass production of recombinant albumin is urgently needed.

Due to this medical importance, attempts have been made to mass produce recombinant albumin using various microbial expression systems by genetic engineering techniques (Goodey, A.R. TIBTECH. 11, 430 (1993)). E. coli or Bacillus microorganisms, which are prokaryotic cells, were expressed as aggregated albumin proteins (Latta et al., Bio / Technology, 5, 1309 (1987)), or their secretion efficiency was extremely low (Saunders). et al., J. Bacteriol. 169, 2917 (1987)). On the other hand, when a yeast, which is a single cell microorganism and a eukaryotic cell, is used as a host, correctly produced albumin has been reported to be efficiently secreted into the cell culture medium. Saccharomyces cerevisiae, a baker's yeast that has traditionally been used as a recombinant protein production host [Sleep et al., Bio / Technology 8, 42 (1990), Okabayashi et al., J. Biochem. 110, 103 (1991), Kang et al., J. Microbiol. Biotechnol., 8, 42, (1998)], as well as the expression of recombinant albumin was secreted by Kluyveromyces lactis and Pichia pastoris, which were developed as a new alternative yeast [Fleer et. al., Bio / Technology 9, 968-975 (1991); Sreekrishan, K. Industrail microorganisms, pp 119-126].

Disadvantages of yeast Saccharomyces cerevisiae as a host cell in recombinant protein production, such as the absence of strong and finely regulated promoters, the need for fed-batch cultures at high concentrations, instability of expression vectors at long fermentation, sugars Several other yeast systems have recently been developed as alternative hosts to compensate for problems such as protein glycosylation (Romanos et al., Yeast 8, 423 (1992)). Among them, Hansenula polymorpha, along with Pichia pastoris, is a magnetized yeast of methanol, which is attracting attention as a novel recombinant protein expression system due to the advantages of high productivity and high concentration culture (Faber et al., Yeast 11, 1331 (1995). They are economical because they can use methanol as an inexpensive raw material as an energy source and a carbon source, and they are relatively economical, such as promoters of methanol oxidase (MOX) or alcohol oxidase (AOX) genes involved in methanol metabolism. It is easy to adjust and has a very powerful promoter, which makes it highly applicable in industrial applications. In addition, high-concentration culture (100-130g DCW / L) is easy, and when transforming, a number of expression target genes are introduced on the chromosome of the host.

These methanol magnetizing yeasts have been reported to date with 22 nm hepatitis surface antigen protein (Janowicz et al., Yeast 7, 431 (1991)), human epidermal growth factor (Clare et al., Gene 105, (1991)), glucoamylase ( Over 20 industrially useful recombinant proteins have been successfully produced, including Gellissen et al., Bio / Technology 9, 291 (1991), and Eurokinase (Tsujikawa et al, Yeast 12, 541 (1996)). The production of the same recombinant protein is often higher than that of Saccharomyces cerevisiae (Buckholz and Gleeson Bio / Technology 9, 1067 (1991); Cregg et al., Bio / Technology 11, 905). 1993). Human serum albumin also produced significantly higher yields than those reported in Saccharomyces cerevisiae using the AOX promoter in Pichia pastoris (Cregg et al., Bio / Technology 11, 905 (1993)). .

The present inventors have made their own efforts to develop Hansenula polymorpha, a methanol magnetizing yeast having the advantages described above, as a general recombinant protein expression system. ) Was cloned (Sohn et al., J. Bacteriol. 178, 4420 (1996)), using which antithrombotic hirudin (Kim et al., Biotechnol. Lett. 18, 417) in Hansenula polymorpha. (1996)), human epidermal growth hormone (Oh et al., Kor. J. Appl. Microbiol. Biotechnol. 22, 477 (1994)) and the plasma protein alpha1-antitrypsin (Kang et al., Yeast 14, 371 (1998)) was successfully established.

In an effort to efficiently express human serum albumin in yeast, the present inventors have expressed human serum albumin (HSA) in Hanshenula polymorpha using Hansenula polymorpha, a methanol magnetizing yeast, as a host cell. A vector suitable for the assembly was assembled. To develop a recombinant albumin expression vector with high expression rate, various vector components such as 5'untranslated region (5'UTR), selection marker, and self-replicating sequence (ARS) are assembled with different expression vectors. The effect on the conversion efficiency and the expression of human serum albumin was investigated, and the transformants introduced into the chromosome were selected and the strains which produced albumin with high efficiency were selected to express recombinant human serum albumin.

The present invention assembles the expression vector using an albumin cDNA comprising a secretion signal of the MOX promoter and albumin itself, and introduced into the yeast Hanshenula polymorpha DL1-L, the assembled expression vector is integrated into the chromosome (integration) The present invention provides a method of producing albumin by preparing a transformant and selecting a transformed strain that produces albumin from high efficiency.

More specifically, self-replicating sequence (ARS), 5'UTR and screening for efficient expression of human serum albumin from albumin cDNA with albumin itself secretion signal using Hansenula polymorpha DL-1 derived MOX promoter The present invention provides a method for producing human serum albumin by assembling several human serum albumin expression vectors having different labels, and selecting the transformants into which these vectors are introduced and selecting the highly efficient expression strain.

1 is a schematic diagram of a method for assembling albumin expression vectors for Hanshenula polymorpha.

2 is a schematic of the 5′UTR of cDNA encoding human serum albumin linked to MOX promoter.

Figure 3 is a photograph showing the results of screening the high-efficiency production strains by culturing Hansenula polymorpha transformed with albumin expression vector on a nitrocellulose disk and Western blot.

Figure 4 is a result of Western blot after culture of the selected yeast strain SDS-polyacrylamide electrophoresis.

Figure 5 is the result of Southern blot to measure the copy number of the yeast strain DNA containing the albumin expression vector.

Figure 6 is the result of Northern blot to measure the degree of gene transcription expression extracted RNA of yeast strain containing albumin expression vector.

Figure 7 is the result of staining after SDS-polyacrylamide electrophoresis of albumin-produced yeast culture for use in N-terminal amino acid analysis of recombinant albumin.

8 is a result of measuring the pI value by electrophoresis of the culture concentrate of the recombinant albumin protein on an isoelectric focusing gel.

The present invention assembles an expression vector using an albumin cDNA comprising a secretion signal of albumin itself and a MOX promoter derived from yeast Hanshenula polymorpha DL-1, and the expression vector is assembled by introducing it into the yeast Hanshenula polymorpha. The present invention provides a method for producing human serum albumin, characterized by expressing human serum albumin with high efficiency by selecting and fermenting and culturing the transformed yeast inserted into the chromosome. In addition, the fermentation culture is characterized in that the transformed yeast is cultured in a medium containing methanol. Albumin cDNA is also a gene isolated from pBlue-HSA1 derived from the cDNA library made from Korean fetal liver cells.

Meanwhile, the expression vector pYHSA12 (KCTC-0503BP) and the expression vector pYHSA12 inserted into the chromosome of the host cell were removed from the albumin-encoding cDNA and 5'UTR (5 'non-toxic region) and albumin initiation codon was directly linked to the MOX promoter. (Integrated) transformed yeast Hanshenula polymorpha DL1-L / T7 (KCTC-0502BP) and transformed yeast Hanshenula polymorpha DL1-L / with the expression vector pYHSA12 integrated into the chromosome of the host cell. T8 (KCTC-0501BP).

Accordingly, the present invention provides a process for assembling expression vectors suitable for expressing human serum albumin using methanol magnetized yeast Hanshenula polymorpha as a host cell, and selecting a yeast transformant containing the expression vector, and from the same, a highly efficient expression strain. And a method for producing human serum albumin from high-efficiency expression strains.

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

(Experimental strain and gene)

The human serum albumin cDNA gene was derived from pBlue-HSA1 [Kang et al., J. Microbiol. Biotechnol., 8, 42, (1998). In addition, as a yeast Hanshenula polymorpha host for the expression of albumin, DL1-L (leu2), a leucine nutritional constitutive strain provided by NPO Biotechnologia (Moscow, Russia) and strain DL1L-uv10 (leu2, obtained by UV irradiation) ura3) and the like. The MOX promoter and MOX terminator of Hansenula polymorpha DL-1 (ATCC-26012) were obtained from pMOX3615 (Biology Research Report BSKG 1050-885-3), which was cloned with 9 kb of chromosomal DNA fragments containing the MOX gene.

(Preparation of new transformed yeast cell line)

Albumin expression vectors were introduced into yeast Hanshenula polymorpha DL1-L or DL1L-uv10 strains by Hill et al. (Hill et al., Nucl. Acids Res. 19, 5791 (1991)) to URA3 + or LUE2 ⁺ . Converted transformants were selected. In the case of the expression vector using the uracil synthetic gene URA3 derived from Saccharomyces cerevisiae, the culture was cultured in a uracil-deficient medium until the transformant was stabilized. Vectors using the APH gene, which has resistance to LEU2 derived from Hanshenula polymorpha and G418 derived from E. coli, were obtained as LUE2 ⁺ transformant first, and then passaged about 50 generations in YPD medium. After stabilization, the cells were plated in different G418 concentrations for secondary screening.

(Medium composition and culture conditions)

As a seed medium, yeast strains were incubated for 16-20 hours using YPG medium (1% yeast extract, 2% Bakto peptone, 2% glycerol), followed by 30 ml expression-induced YPM medium (yeast extract 1). %, Bacterium peptone 2%, methanol 2%) was inoculated at 1% in a 250 ml baffled flask (1%) was incubated for 24 hours shaking. During methanol feeding, methanol was added up to 72 hours every 12 hours after incubating in YPM medium for 24 hours so that the methanol concentration in the medium was 2%. Incubation temperature was maintained at 37 ℃.

(Western Blot Analysis)

Cells and culture supernatants were separated from the cell culture by centrifugation, and the culture supernatants were electrophoresed on SDS-polyacrylamide gels by Laemmli, Nature 227, 680 (1970) method, followed by Towbin (Towbin, Proc. Natl. Acad. USA, 76, 4350 (1979)) was attached to the nylan membrane and reacted with a polyclonal antibody against human serum albumin (Sigma). Secondary reaction was performed with an antibody against anti-rabbit Ig (Sigma) attached to alkaline phosphatase and the albumin expression level was analyzed by color reaction by alkaline phosphatase. The amount of the expressed albumin was quantified by reading the sensitivity of the protein band in the western blot using a plasma albumin purchased from Sigma, USA with a densitometer (Image analysis system, Bio-Rad, Model GS-700).

Southern blot analysis

In order to analyze the pattern and copy number of the albumin expression vector on the chromosome of yeast Hanshenula polymorpha DL1-L, DNA was extracted from transformants cultured for 24 hours in mixed medium YPD medium. After cleavage with enzyme, it was performed by the method of Sambrook et al. (Molecular cloning Cold Spring Harbor Laboratory Press, 1989). Probes were prepared by labeling the 1.5 kb MOX promoter gene with digoxigenin using a non-radioactive DNA labeling and detection kit. To determine the number of multiple introduction of albumin expression vectors, the sensitivity of the DNA band was determined by reading using a densitometer.

(Northern blot analysis)

After recovering the transformants cultured in the expression induction medium for about 8-15 hours, RNA was extracted by hot phenol method (Elion and Warner, Cell 39, 663 (1984)) and Sambrook et al. (Molecular Cloning Cold Spring Harbor Laboratory Press) , 1989). The probe used the MOX promoter gene labeled with the non-radioactive Digoxygenin prepared above.

Hereinafter, the present invention will be described in detail with reference to Examples and Examples. However, these examples do not limit the scope of the present invention.

Example 1 Preparation of Human Serum Albumin Expression Vector Using MOX Promoter

In order to induce the expression of human serum albumin in Hanshenula polymorpha, we use the MOX promoter known as a potent and controllable promoter.Saccharomyces sere as a secretion signal sequence for secreting the expressed albumin into yeast cultures. The secretion sequence of human serum albumin (hereinafter referred to as ″ HSA ″) itself (Vang et al., J. Microbiol. Biotechnol. 8, 42 (1998)), in which very high secretion efficiency was confirmed in Vichy was used. Several expression vectors were assembled to examine the effect of the selection marker and the presence of ARS on the conversion rate and multiple expression vector introduction rates. In addition, in order to examine the effect of 5'UTR of HSA cDNA introduced into HSA expression cassette on HSA expression in Hansenul or polymorph, HSA 5'UTR was removed and the HSA expression cassette directly connected to MOX promoter was assembled.

1) Assembly of YIP-HSA

To connect the MOX terminator to the HSA cDNA, a 0.5 kb BglII / EcoRV DNA fragment containing the MOX terminator was first obtained from pMOX3615 and cloned into the BamHI and SmaI sites of pBluescriptKS (Stratagene) to assemble pBKS-Tmox (I). In pBKS-Tmox (I), the EcoRI and HindIII sites of multiple cloning sites were removed by Klenow fragment treatment to assemble pTmox (II). pTmox (II) was cleaved with NotI, then treated with Krynau, and again cleaved with XhoI to obtain a 0.5 kb MOX terminator, and inserted into the Krynach-treated XhoI site and HindIII site of pBlue-HSAI to complete pHSA-Tmox. . pHSA-Tmox was cleaved with EcoRI and ClaI to obtain an HSA cDNA fragment (2.5 kb) linked to the MOX terminator and linked with the 1.5 kb SalI / EcoRI MOX promoter derived from pMOX3615 to the vector YIP5 (Struhl et al., Proc. Natl. Acad. YIP-HSA was assembled by insertion into SalI and EcoRI sites of Sci. USA 76, 1035 (1979)). Thus, YIP-HSA contains a 3.5 kb HSA expression cassette in which the terminator is connected to the MOX promoter of Hansenula polymorpha in HSA cDNA with 5′UTR (FIG. 1).

2) Assembly of pUR-HSA

PCEU-EGF was cleaved with NotI, kleenau fragmented and then XbaI to assemble an HSA expression vector containing HARS36 (Sohn et al., J. Bacteriol. 178, 4420 (1996)) that induces vector multiduction. PUR-HSA was assembled by linking the 3.5 kb HSA expression cassette obtained by cleaving YIP-HSA with SalI and digesting with Krinau and digesting with XbaI in the 7 kb vector skeleton obtained by (Fig. 1).

3) Assembly of pDLMOX-G418HSA

2 kb EcoRI / HindIII HSA cDNA obtained from pBlueHSA1 was synthesized by pDLMOX-HIR (Kang et al., Yeast 14, 371 (1998)) to assemble an expression vector of Hansenula polymorph LEU2. The expression vector pDLMOX-HSA was assembled by HSA cDNAI linked to a terminator derived from a MOX promoter and a gene not yet analyzed. PDLMOX-G418HSA was assembled by inserting a 1.3 kb G418 resistance gene (kan ^r ) derived from pUC4K (Pharmacia) into the SnaBI site of the vector (FIG. 1).

4) Assembly of pYHSA12

Polymerase chain reaction (PCR) was used to directly connect 5'UTR-depleted HSA cDNA to the MOX promoter without using restriction enzymes. Two oligonucleotides MOX / HSA-33mer (5'-CTAAAGTACAAAAACAAAATGAAGTGGGTAACC-3 ') (initiation codon of HSA gene underlined) and HSA (AflIII) -19mer (5'-GCTGACTCATCAGCAACAC-3') DNA fragments containing about 250 bp of HSA N-terminal portion directly linked to the 5'UTR (non-translated region) of the MOX promoter from pBlue-HSAI were directly synthesized. In a second step, a 1.5 kb SalI / EcoRI MOX promoter DNA fragment derived from pMOX36159 was previously reported from pBMOX8 inserted into pBluescriptKS (Sohn et al., J. Microbiol. Biotechnol. 2, 65 (1993)). A 1.5 kb sized MOX promoter DNA fragment was synthesized using reverse primer (5'-AACAGCTATGACCATG-3 ') and MOX-19mer (5'-CATTTTGTTTTTGTACTTT-3'). After mixing the DNA fragments synthesized in the first step and the second step together, 1.8 kb DNA fragments in which the HSA cDNA N-terminus is connected to the MOX promoter by PCR using MOX / HSA-33mer and reverse primers After synthesis, it was inserted into pT7Blue (Novagen) to assemble pT7Blue / PmoxHSA247. The PCR was performed at 95 ° C. 30 sec, 55 ° C. 1 min, 72 ° C. 3 min, and 35 times conditions. Changes in nucleotide sequence that could occur during PCR were confirmed by nucleotide sequence analysis. A 1.8 kb MOX promoter and HSA cDNA N-terminus cleaved from pT7Blue / PmoxHSA247 with AflIII and SalI were used to obtain DNA fragments. HSA cDNA with 5'UTR removed by assembling with AflIII and EcoRI DNA fragments assembled a 3.5 kb HSA expression cassette linked to the MOX promoter and terminator of Hansenula polymorpha DL-1. NotI restriction enzyme recognition site of vector pGLG61 (submitted to Sohn JH et al, Yeast) containing the Hansenula polymorpha LEU2 gene and the foreign gene G418 resistance gene (kan ^r ) cassette as the selection marker. Cloned into pYHSA12 (FIG. 1). Thus, pYHSA12 has an albumin expression cassette in which the HSA cDNA from which the 5′UTR was removed was linked to the MOX promoter (FIG. 2).

Recombinant expression vector pYHSA12 prepared by the above method was deposited on July 14, 1998 to the Biotechnology Center in the Korea Institute of Science and Technology under the accession number KCTC-0503BP.

(Example 2) HSA expression transformant secured

1) Primary screening using nutritional urine screening label

When transforming DL1-L (leu2) or DL1L-uv10 (ura3 leu2) strains with the expression vectors, YIP-HSA, an expression vector without ARS, was too low to obtain a transformant, but HARS36. In the whole expression vector pUR-HSA, URA3 + transformants were obtained at a relatively high rate, and through the stabilization of the expression vector, strains in which HSA expression vectors were introduced into the chromosome of the host in several copies were obtained. However, since pUR-HSA uses URA3 of Saccharomyces cerevisiae, which has low expression efficiency in Hanshenula polymorpha, it has a lower transformation rate compared with LEU2 of Hanshenula polymorpha as a selection label. Very much time and labor was spent stabilizing the transformants. As in the case of pDLMOX-G418HSA, when only Hansenula polymorph LEU2 is used as a marker, most transformants are introduced into the host chromosome by only one or two copies, whereas pYHSA12 is a G418 resistance gene (kan) expressed in Hansenula polymorpha. ^r ) The LEU2 + transformant obtained by having the expression cassette was subjected to secondary screening by G418 concentration, and the multi-introduced strains were selected therefrom.

2) HSA expression strain selection by colony blot

The obtained transformants were grown on a YPG plate for about 15-20 hours, and then attached to a nitrocellulose membrane. The transformants were transferred to YPM medium containing methanol and incubated at 37 ° C. for 24 hours. Cells and other debris on the nitrocellulose membrane were washed three times with buffer (20 mM Tris-HCl, pH 7.5, 500 mM) and subjected to western blot using albumin polyclonal antibody. The process was selected three strains finally showing a dark color reaction (Fig. 3). Selected strains were incubated for 24 hours in a 15 ml test tube containing YPD, the culture supernatant was subjected to SDS-polyacrylamide electrophoresis, and the degree of HSA expression was reviewed by Western blot (FIG. 4).

Example 3 Expression of HSA in Flask Culture

When Hansenula polymorpha produces recombinant protein under the control of MOX promoter, it uses methanol as a source of induction for expression of MOX promoter as well as a carbon source required for growth of strain itself for albumin expression. Methanol was fed at regular time intervals to induce sustained expression. In the case of baker's yeast Saccharomyces cerevisiae, the pH of the medium was acidified as the incubation time progressed, and if only a carbon source such as galactose was continuously supplied, the pH of the medium was reduced more rapidly and the expression of HSA protein was accelerated. (Kang et al., J. Microbiol. Biotechnol. 8, 42 (1998)). On the other hand, in case of Hanshenula polymorpha, even though the culture was continued for about 3 days in YPM medium containing methanol, the pH of the medium was increased to about 7.9, and there was no rapid decomposition of the expressed HSA. However, when methanol was continuously supplied at 12-hour intervals, the amount of decomposition products was detected, although the amount of HSA of the full size (66 KDa) in the medium was not changed significantly compared with the case where methanol was not supplied. This suggests that the total amount of HSA produced was more during the continuous supply of methanol, but such a culture method induced the action of any protease so that the HSA was degraded and the amount of HSA present in the medium was not increased. When comparing the expression of albumin by two culture methods, the continuous growth of methanol increased cell growth, but the value of albumin expression per unit cell decreased rather (Table 1). Therefore, if studies on culture conditions to prevent HSA degradation, such as Saccharomyces cerevisiae, can be performed in Hanshenula polymorpha, the current yield (100 mg / L in flask culture) can be further improved. .

Effect of Methanol Supply on HSA Expression in Hansenula Polymorpha OD ₆₀₀ pH ^a HSA ^b (μug / ml) Specific productivity (μg / OD ₆₆₀ ) YPM ^c 49 7.9 100 2.04 YPM + M ^d 81 7.8 100 1.23

^a The final pH value of the culture supernatant.

^b Albumin expression was measured for cell supernatant cultured for 3 days.

^c yeast extract (1% w / v), peptone (2% w / v), methanol (2% w / v)

^d Methanol (2% w / v) feeding was fed every 12 hours after 24 hour incubation.

(Example 4) Expression vector introduction water analysis by Southern blot

Southern blot analysis of the selected transformants yielded transformants in which the expression vector pUR-HSA was introduced into the host chromosome up to about 7 copies, and in the case of pYHSA12, the concentration of G418 was high. It was found that a transformant in which HSA expression vector copy numbers were introduced from 1 to 6 was obtained. The site of introduction of these vectors is largely divided into two classes, sometimes introduced into MOX genes or unknown gene positions (FIG. 5).

Example 5 Comparative Analysis of HSA Expression Efficiency in Hansenula Polymorpha Transgenic Lines

The obtained transformants were cultured to analyze the amount of HSA protein by Western blot and the amount of HSA mRNA by Northern blot to compare HSA expression efficiency. Strain 827d-5, in which the expression vector pUR-HSA was introduced in about 7 copies, had a 7-fold increase in HSA mRNA expression in proportion to the increase in the number of copies compared to strain 827d-8, which was introduced in 1 copy. The increase was about 5 times (Table 2). As a result of comparative analysis of Northern blot of transformants introduced with pUR-HSA and transformants with 5'UTR removed from HSA cDNA, the removal of 5'UTR was not relevant to HSA mRNA expression. Although it did not affect (FIG. 6), it was confirmed that the HSA protein expression efficiency was increased by about 8-10 times. However, the transformants with the expression vector pYHSA12 did not show an increase in HSA protein production according to the increase in the number of HSA expression vectors, which was analyzed because there was no increase in the amount of HSA mRNA expression according to the increase in the number of vectors. 6). Table 2 shows the total number of HSA expression vector introduced, HSA mRNA expression amount and HSA protein secretion expression amount of each transformant.

Southern, Northern, and Western blot analysis of Hanshenula polymorphs Transformant Expression vector HSA-5'UTR Southern Northern Western ^b Insertion Area Copy number mRNA expression Protein expression amount (mg / L) u10-827d-8 pURHSA + uk ^a One ++ 5 u10-827d-5 〃 + uk 7 +++++++ 10 to 20 DL1-L / T7 pYHSA12 - mox One ++ 40-50 G3T7 〃 - mox 2 ++ 40-50 DL1-L / T8 〃 - mox 2 ++ 40-50 G4T9 〃 - mox 3 ++ 40-50 G0.8T3 〃 - uk One + 20-30 G4T13 〃 - uk 2 ++ 40-50 G2T7 〃 - uk 3 + 20-30 G3T5 〃 - uk 6 + 20-30

^a unknown

^b After 24 hours of incubation in a 250 ml baffle flask, the expressed albumin of the culture supernatant was quantified.

Hansennura polymorpha DL1-L / T7 strains with the highest performance among the transformed strains were deposited with KCTC-0502BP No. KCTC-0502BP at the Biotechnology Center of the Korea Advanced Institute of Science and Technology on July 14, 1998. L / T8 strain was deposited with accession number KCTC-0501BP.

Example 6 Recombinant Albumin Characterization

1) N-terminal amino acid sequence analysis of expression-secreted recombinant albumin

Most of the recombinant albumin expressed and expressed in the transformants had the same size (66 kDa) as albumin isolated from human plasma, but were observed in baker's yeast Saccharomyces cerevisiae as the incubation time increased. HSA degradation products of size kDa were detected and in addition, other degradation products of size 52 kDa were also detected (FIG. 7). SDS-electrophoresis of the culture supernatant concentrated using YM10 membrane (Amicon), transferred to PVDF membrane for amino acid analysis, stained with Ponso-S, corresponds to the protein band corresponding to albumin 66 kDa and the degradation product of about 45 kDa N-terminal amino acid sequence was analyzed by separating the bands. As a result, both proteins showed an N-terminal amino acid sequence starting with D-A-H-K-S, similarly to plasma albumin, thereby confirming that albumin secretion signals were correctly cleaved in Hanshenula polymorpha.

2) Analysis of isoelectric values (pI values)

In order to analyze the isoelectric point of the recombinant albumin, the cultured supernatant of the transformed strain was electrophoresed in a pI 3-7 undenatured isoelectric focusing gel sold by Novex, USA. It was soaked in Tris-glycine solution for about 30 minutes, blotted onto PVDF membrane, and Western blot was used to determine the pI value. As an isoelectric standard protein, a product having a value of pI 4.45-9.6 manufactured by Bio-Rad, USA was used. The isoelectric value of recombinant albumin expressed in Hanshenula polymorpha was found to be around pI 4.8, similarly to serum albumin (Sigma) (FIG. 8).

The effect of the present invention is to cultivate the recombinant Hansenula polymorpha mutants selected from the high-efficiency production mutants transformed with the assembled HSA expression vector in a medium containing methanol, thereby mass-producing expression-recombinant recombinant human serum albumin. It provides a way to do it. Therefore, the present invention is a very useful invention in the pharmaceutical industry in which the demand for albumin is rapidly increasing.

Claims (6)

An expression vector was assembled by using an MOX promoter derived from yeast Hanshenula polymorpha DL-1 and albumin cDNA containing the secretion signal of albumin itself, and then introduced into the yeast Hanshenula polymorpha. Method for producing human serum albumin characterized by expressing human serum albumin with high efficiency by screening and fermenting cultured transformed yeast The method for producing human serum albumin according to claim 1, wherein the fermentation culture comprises culturing the transformed yeast in a medium containing methanol. The method of claim 1, wherein the albumin cDNA is a gene isolated from pBlue-HSA1 derived from a cDNA library made from Korean fetal hepatocytes. Expression vector pYHSA12 (KCTC-0503BP) with 5'UTR (5 'non-toxic region) removed from albumin-encoding cDNA and albumin initiation codon directly linked to MOX promoter Transformational yeast Hanshenula polymorpha DL1-L / T7 with expression vector pYHSA12 integrated into chromosome of host cell (KCTC-0502BP) Transformed yeast Hanshenula polymorpha DL1-L / T8 (KCTC-0501BP) with the expression vector pYHSA12 integrated into the host cell chromosome