KR100697308B1 - A novel Catalase promoter and the method of producing recombinant proteins using the same - Google Patents

Abstract

The present invention relates to a catalase promoter nucleic acid molecule and a method for producing a protein of interest using said promoter.

Hansenula polymorpha, catalase, promoter, expression vector, recombinant protein

Description

A novel Catalase promoter and the method of producing recombinant proteins using the same}

1 is an expression plasmid construct constructed to induce recombinant protein expression from Hanshenula polymorpha to catalase (CAT) promoter. Figure 1a is a plasmid configured to express glucose oxidase (GOD) derived from Aspergillus niger , Figure 1b is a plasmid for expressing human serum albumin (hSA), Figure 1c is a green fluorescent protein (GFP) The plasmid constructed for expressing) is shown.

Figure 2 shows the results of measuring the activity of GOD in a plate medium after expression of GOD using the CAT and MOX promoter in Hansenula polymorpha. A shows the result of measuring the activity of GOD expressed by culturing the transformant in YPM complex plate medium containing methanol as carbon source, and B is the activity of GOD expressed after culturing the transformant in YPD complex plate medium using glucose as carbon source. The result of the measurement is shown.

Figure 3 shows the result of comparing the amount of enzymes for cell growth by carbon source by expressing GOD in liquid culture using CAT and MOX promoters from Hanshenula polymorpha. Black indicates glucose medium and white indicates methanol medium.

4 is a result of qualitative analysis of hSA expression characteristics by staining proteins by silver staining after developing hSA expression using Hansenula polymorpha using CAT and MOX promoters. A is a result of comparing the expression characteristics of Hanshenula polymorpha MOX and CAT promoter, B is the result of examining the effect of 5-UTR on the expression of hSA by CAT promoter.

5 is a result of examining the expression characteristics of GFP by a fluorescence microscope using the CAT and MOX promoters from Hanshenula polymorpha and quantitating the expression characteristics of GFP by fluorescence absorbance.

The present invention relates to a catalase promoter nucleic acid molecule and a method for producing a protein of interest using the same.

Hansenula polymorpha, one of the methanol magnetizing yeasts, has a high cell growth rate, strong promoters and easy introduction of foreign genes, thereby replacing the production of recombinant protein Saccharomyces cerevisiae . It is highly regarded as an industrial microorganism (Gleeson et al. Yeast, 4, 1 (1988); Janowicz et al. Yeast, 7, 431 (1991); Romanos et al. Yeast, 8, 423 (1992); Faber et al. Yeast, 11, 1331 (1995). Recently, mass production of hepatitis B vaccine using it has been commercialized in Argentina, Brazil and Korea (Hansenula polymorpha: Biology and Applications, Gellison ed. P.186, Wiley-VCH Verlag GmbH, Weinheim (2002)). The system has been evaluated and applied to the production of various recombinant proteins. In addition, studies on the production of novel and generic recombinant proteins adapted for human glycoprotein production using an efficient glycosylation process of Hanshenula polymorpha (Kang et al. KR2002-37717; Kang et al. KR2003-43544; Kang et al. PCT / KR03 / 01285; Kang et al. KR2004-06352; Kang et al. PCT / KR04 / 001819).

Promoters for the expression of foreign genes from Hanshenula polymorpha include methanol oxidase (MOX) related to methanol metabolism (Ledeboer et al. Nucleic Acids Res. 13, 3063 (1985)), dihydroxyacetone synthesis Enzyme (Dihydroxyacetone synthase) (Janowich et al. Nucleic Acids Res. 13, 3042 (1985)) and formic acid dehydrogenase (FMDH) (Hollenberg et al. EPA0299108 (1987)), among others, have been developed. The promoter of oxidase is very powerful and precisely controllable, so it is widely used in recombinant protein production. These methanol metabolism-derived promoters are generally highly expressed in the presence of methanol, whereas expression is inhibited in the presence of other carbon sources such as glucose, glycerol or ethanol, thus supplying methanol as the main carbon source for cell culture for recombinant protein production. Done. However, formaldehyde, produced by methanol metabolism, has a fatal effect on cell survival, so it is necessary to supply methanol very carefully to minimize this risk. Because of this, the growth rate of the cells is very slow and requires a long incubation time, FMDH promoter has been used as an alternative to the MOX promoter when the expression of the promoter at low concentration glucose and glycerol supply is easier.

Hanshenula polymorpha is unlike other methanol magnetizing yeasts, especially Pichia pastoris , which has matured due to its high industrial applicability. Represents a form of suppression-unwinding-induction, and thus there is a disadvantage in that induction of a methanol metabolism related promoter does not occur without methanol), and since glucose inhibition is easily solved, there is an advantage of replacing methanol induction.

Glucose inhibition is a common phenomenon seen in microorganisms and has been reported to inhibit the expression of metabolic enzymes related to carbon sources other than glucose in the presence of glucose at the transcriptional level, and methanol metabolism and peroxysome in the case of Hanshenula polymorpha. It is known that most of the enzymes involved are inhibited at the transcriptional level, in particular methanol oxidase is very strongly inhibited. On the other hand, formic acid dehydrogenase is related to methanol metabolism, but low concentrations of glucose have been reported to be easily suppressed unlike methanol oxidase. Thus, recombinant protein production systems using FMDH promoters are preferred in the industrial field.

In addition, the amine oxidase (Weydemann et al. Appl. Microbiol. Biotechnol. 44, 377-385 (1995)) promoters involved in the metabolism of primary amines, the YNT1, YNR1 and YNL1 promoters (Perez et. al. Biochem. J. 321, 397-403 (1997); Avila et al. FEBS Lett. 366, 137-142 (1995); Brito et al. Biochem. J. 317, 89-95 (1996)), phosphorylation In connection with the acid phosphatase (PHO1) promoter (Phongdara et al. Appl. Microbiol. Biotechnol. 50, 77-84 (1998)), the glyceride 3-phosphate dehydrogenase (GAP) promoter (Heo et al. FEMS Yeast) Res. 4, 175-184 (2003); 237979, Republic of Korea; and trehalose 6-phosphate synthase (Reinder et al. J. Bacteriol. 181, 4665-4668 (1999) in relation to the sugar pathway. )) are being developed, in recent years, the plasma membrane ^{H + -ATPase (plasma membrane H +} -ATPase;. PMA1) promoter (Cox et al Yeast, 16, 1191-1203 (2000) and translation elongation factor 1a (trans lation elongation factor 1a; TEF1, TEF2) promoters (Terentiev et al. J. Ind. Microbiol. Biotechnol. 31, 223-228 (2004)) have been developed. These expression rates, except for the FMDH promoter and some promoters, do not show strong expression capacity as a replacement for the MOX promoter, and are mostly protected by patents, and thus are limited in industrial use having a unique expression system.

Therefore, there is a need for the discovery of novel promoters that can be easily derived from glucose or glycerol as well as methanol while having strong promoter activity in place of existing MOX and FMDH.

Under this background, the inventors found that the promoter of catalase enzyme according to the present invention is an efficient promoter that induces expression in the presence of low concentrations of glucose or glycerol as well as methanol, and it is possible to effectively produce recombinant protein using the promoter. It confirmed and completed this invention.

One object of the present invention is to provide a promoter nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary to the nucleotide sequence.

Still another object of the present invention is to provide an expression vector comprising the promoter nucleic acid molecule.

Still another object of the present invention is to provide a host cell transformed with the vector.

Still another object of the present invention is to transform a vector comprising a nucleic acid sequence operably linked with the promoter nucleic acid molecule and the nucleic acid molecule encoding a protein of interest to a host cell, the presence of methanol, glucose, glycerol or a combination thereof. It is to provide a method for producing a target protein comprising the step of inducing the expression of the target protein, and the step of separating and purifying the target protein.

In one embodiment, the invention relates to a catalase promoter nucleic acid molecule.

In a more preferred aspect, the present invention relates to a promoter nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary to a nucleotide sequence.

As used herein, the term “promoter” refers to a non-translated nucleic acid sequence upstream of a coding region that contains a binding site for polymerase and has a transcription initiation activity to mRNA of a promoter subgene. The promoter according to the present invention is a promoter of a natural catalase (CAT) gene isolated from the genome of Hanshenula polymorpha, and has been deposited with GeneBank as accession number AY748852 with the nucleotide sequence of SEQ ID NO: 1. The CAT promoter of the present invention is an inducible promoter requiring a specific signal, methanol, glucose or glycerol acts as an inducer.

Catalase is an enzyme that catalyzes the conversion of hydrogen peroxide produced by methanol oxidation into water and oxygen in methanol metabolism. Catalase studies are related to the oxidative stress of cells and metabolism of peroxisome. Report on the study (Hansen and Roggenkamp, Eur. J. Biochem. 184, 173-179 (1989); Weiser et al. J. Biol. Chem. 266, 12406-12411 (1991); Fukamatsu et al. Plant Cell Physiol 44, 982-989 (2003); Horiguchi et al. J. Bacteriol. 183, 6372-6383 (2001)), but no studies on catalase promoter identification have been reported.

In the promoter of the present invention, the expression of catalase was reduced by 12.48 by the glucose and methanol carbon sources through a hybridization experiment of the microarray slides prepared for all genes of Hanshenula polymorpha and the mRNA of the carbon source treated samples of glucose and methanol. Primary selection in fold increase. PCR of chromosomal DNA obtained from Hanshenula polymorpha A16 strain based on genome sequence information (Ramezani-Rad et al. FEMS Yeast Res. 4, 207-215 (2003)) for Hanshenula polymorpha RB11 strain The DNA fragment of 1,252 bp containing the Hansenula polymorpha catalase promoter was analyzed, and the sequencing was performed. Then, the activity level of the CAT promoter was measured using the existing MOX promoter, glucose oxidase (GOD), and recombination. The expression levels of human serum albumin (hSA) and Green Fluorescent Protein (GFP) were compared and analyzed. Promoter activity is determined by linking downstream of the promoter to express a gene encoding a quantifiable protein (reporter gene) and measuring the amount of mRNA or protein expressed in the presence of an inducer. As a result, CAT promoter is not only induced expression by methanol carbon source but also excellent expression induction in presence of low concentration of glucose carbon source, so it can be effectively used for recombinant protein production in fermentation process using glucose as carbon source, expression of CAT promoter Unlike the MOX promoter, the modulus was strong at the early stage of induction, which prevented denaturation of the expressed protein by prolonged culture by shortening the cell culture time.

The sequence encoding the promoter of the present invention can be modified. One of ordinary skill in the art will appreciate that the sequences that maintain at least 70% homology by these artificial modifications are equivalent to those derived from the base sequences of the present invention as long as they retain the promoter activity for gene expression desired in the present invention. Will be easy to understand.

In the present invention, the term “homology” refers to the same degree as that of a wild type nucleic acid sequence, and the comparison of homology refers to the degree of homology between two or more sequences using a comparison program that can be visually or easily purchased. Can be calculated as (%). Preferably, the nucleic acid sequence encoding the promoter region of SEQ ID NO: 1 of the present invention is preferably at least 70%, more preferably at least 80%, even more preferably at least 90% identical.

In addition, promoters of the invention include variants having a promoter nucleic acid sequence in which one or more nucleic acid bases have been modified by substitution, deletion, insertion, or combination thereof, so long as they retain promoter activity. Promoter nucleic acid sequences may induce variations in the sequence by deletion, insertion, non-conservative or conservative substitutions, or a combination thereof. Such sequence variation may exhibit the same activity as the natural promoter, but preferably, the function of the promoter may be improved to suit the purpose, such as a promoter having increased activity or a promoter having increased specificity for an inducing agent.

Nucleic acid molecules encoding all of the above categories of promoters are provided as promoter components of an expression vector that induces the expression of a gene of interest, and modifications of various vectors using the promoter are included in the scope of the present invention.

In another aspect the invention relates to an expression vector comprising said promoter nucleic acid molecule.

As used herein, the term “expression vector” refers to a gene construct including essential regulatory elements such as a promoter to express a target protein in a suitable host cell. Expression vectors associated with the present invention are vectors in which the promoter is a CAT promoter and includes viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, yeast vectors, or adenovirus vectors, retrovirus vectors, adeno-associated virus vectors. The promoter is operably linked to induce expression of the gene of interest and the vector may be in an integrated form into the genome of the host cell.

In the present invention, "operably linked" refers to a functional link between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. It can be prepared using genetic recombination techniques well known in the art, site-specific DNA cleavage and ligation using enzymes generally known in the art and the like.

As used herein, the term “regulatory element” refers to a non-translated nucleic acid sequence that assists or influences the enhancement of transcription, translation or expression of a nucleic acid sequence encoding a protein. Expression vectors of the present invention essentially include a CAT promoter as a regulatory element, and expression control sequences that may affect the expression of proteins such as initiation codons, termination codons, polyadenylation signals, enhancers, membrane targeting or secretion It may include a signal sequence for.

Polyadenylation signals increase the stability of transcripts or facilitate cellular transport. Enhancer sequences are nucleic acid sequences that are located at various sites in the promoter and increase transcriptional activity as compared to the transcriptional activity by the promoter in the absence of the enhancer sequence. The signal sequence includes PhoA signal sequence and OmpA signal sequence when the host is Escherichia spp., And α-amylase signal sequence and subtilisin signal sequence when the host is Bacillus sp. In the case of MFα signal sequence, SUC2 signal sequence, etc., if the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecular signal sequence, etc. may be used, but is not limited thereto.

In addition, when the vector is a replicable expression vector, it may include a replication origin, which is a specific nucleic acid sequence from which replication is initiated.

In addition, the vector may include a selection marker. The selection marker is for selecting cells transformed with the vector, and markers conferring a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins can be used. Since only cells expressing a selection marker survive in an environment treated with a selective agent, transformed cells can be selected. Typical selection markers used in yeast include β-lactamase gene, tetracycline resistance gene and G418 gene, which are resistant to ampicillin and tetracycline, respectively. Further, comprise yeast ura3-52, his3- △ 1, leu2- △ 1, △ 1 trp1-, lys2-201, such as the nutritional URA3, HIS3, LEU2, TRP1 or LYS2 marker, such that I can be used in the configuration strain Can be.

The nucleic acid sequence encoding the target protein into the vector is inserted into the flame under the promoter and expressed. The target protein that can be inserted into the vector is not particularly limited, but medical and industrially useful target proteins include hormones, cytokines, enzymes, coagulation factors, transport proteins, receptors, regulatory proteins, structural proteins, transcription factors, antigens, antibodies, and the like. have.

Specific examples of the target protein include thrombopoietin, human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferon, interferon receptors, colony stimulating factors, glucacon-like peptides (GLP-1, etc.). ), G-protein-coupled receptors, interleukins, interleukin receptors, enzymes, interleukin binding proteins, cytokine binding proteins, macrophage activators, macrophage peptides, B cell factor, T cell factor , Protein A, allergic inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growth factor, α-1 antitrypsin, albumin, α-lactalbumin, apolipoprotein-E, Erythropoietin, high glycated erythropoietin, angiopoietin, hemoglobin, thrombin, thrombin receptor active peptide, thrombomodulin, blood factor Ⅶ, Ⅶa, , Ⅸ, and XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet Derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, bone formation growth factor, bone formation promoting protein, calcitonin, insulin, atriopeptin, cartilage inducer, elkatonin, connective tissue activator, Tissue factor pathway inhibitors, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenal cortex hormone, glucagon, cholecystokinin, Pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin, US Orstatin, receptors, receptor antagonists, cell surface antigens, virus-derived vaccine antigens, monoclonal antibodies, polyclonal antibodies, and antibody fragments.

In another aspect, the invention relates to a host cell transformed with the vector. Host cells transformable with the vector include Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis or star. Prokaryotic host cells such as Staphylococcus are but are not limited to these. In addition, fungi (eg Aspergillus), yeast (eg Pichia pastoris, Saccharomyces cerevisiae) Cells derived from higher eukaryotes, including lower eukaryotic cells such as Schizosaccharomyces and Neurospora crassa, insect cells, plant cells, mammals and the like, can be used as host cells. Preferably, yeasts, among them, the genus of methylated yeast, the genus Hansenula, Pichia, Candida or Torulopsis, are more preferred.

The method of introducing the vector of the present invention into the cell includes any method of introducing nucleic acid into the cell, and may be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, retroviral infection, microinjection, PEG, DEAE-dextran, Cationic liposome method, lithium acetate-DMSO method, and the like, and in the specific embodiment of the present invention, lithium acetate-DMSO method is used.

In another embodiment, the present invention provides a method for preparing a host cell, comprising transforming a vector comprising a nucleic acid sequence operably linked with the promoter nucleic acid molecule and a target protein into a host cell, methanol, glucose, glycerol or a combination thereof. Inducing the expression of the target protein in the presence of the present invention, and a method for producing the target protein comprising the separation and purification of the target protein.

Cell culture is carried out according to well-known methods, and conditions such as the culture temperature and time, the pH of the medium, the type of inducer, the time of administration of the inducer, and the dosage can be appropriately adjusted.

Proteins are subjected to conventional biochemical separation techniques, such as treatment with protein precipitants (salting), centrifugation, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography. And a variety of chromatography such as affinity chromatography can be used. In order to separate proteins of high purity, these are usually used in combination.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only for the purpose of illustrating the present invention, and the scope of the present invention is not limited only to the following examples.

Example 1 Analysis of Hanshenula Polymorpha Catalase Expression Using Microarrays

Random sequencing tags of Hansenula polymorpha CBS 4732 (http://pedant.gsf.de/cgi-bin/wwwfly.pl?Set=Pichia_ angusta_CBS_4732_RST & Page = index) for Hansenula polymorpha DNA microarray analysis. Primers were prepared for 1,202 pairs of Hansenula polymorpha ORF. Each primer was 18 mer in length and was configured to have 5-aminolink in the forward primer to direct the microarray.

The entire genes for constructing the Hansenula polymorpha microarray were obtained by obtaining the genomic DNA of the Hansenula polymorpha A16 wild type strain according to the method of Holm et al. Gene, 42, 169-173 (1986). 96-well polymerase chain reaction (PCR) using primers prepared above (reaction volume 100 μl; 8 μg / ml chromosomal DNA, 1 pmole / μl primer set, 50 μl 2X premix Taq DNA polymerase) Got as. The PCR reaction product was separated, purified and concentrated in a buffer solution at a concentration of 100 ng / μl and transferred to 384-well using a ProSys5510 microarray (Cartesian Technologies, Irvine, CA, USA) using a super aldehyde slide glass (super-aldehyde). Slide glass, CEL associates, Houston, TX, USA) was spotted and washed with an aqueous solution of sodium borohydride to complete the Hansenula polymorpha microarray slide.

In order to examine the gene expression characteristics of Hanshenula polymorpha against glucose and methanol carbon sources using the Hanshenula polymorpho microarray, the liquid liquid medium YPD-2 (Bacto) was prepared using the Hanshenula polymorpho A16 strain as a carbon source. YPM-1 (Bacto yeast extract 1%, Bacto peptone 2%) using methanol as carbon source after incubation in exponential growth phase (O. D 0.8 ~ 1.0, 600 nm) in yeast extract 1%, Bacto peptone 2%, glucose 2%) , methanol 1%) cells were cultured in a complex liquid medium and centrifuged to obtain total mRNA by Hot phenol method (Elion and Warner, Cell, 39, 663-673 (1984)). In addition, to examine the gene expression pattern of Hanshenula polymorpha against oxidative stress, 1 mM of menadione bisulfite was treated for 2 hours in the above culture conditions to induce artificial oxidative stress. Then, the cells were recovered in the same manner as above to obtain mRNA.

These mRNAs were labeled with fluorescent dyes by direct labeling and indirect labeling. For direct labeling, the method of Hegde et al. (Hegde et al. Biotechniques, 29, 548-562 (2000)) was applied. 100 μg of mRNA sample and 2 μg of oligo-dT were dissolved in 24 μl RNAse-free purified water, incubated for 10 minutes at 70 ° C., and then cooled on ice. 5X reverse transcriptase reaction solution, 0.1 M DTT, 10X dNTP (5 mM d (AGC) TP, 2 mM TTP), labeled fluorescent nucleotides (1 mM Cy3 ^TM -dUTP / Cy5 ^TM -dUTP, Amersham Bioscience, Piscataway, NJ, USA), RNAsin (RNAse inhibitor), and 200 U Superscript ^TM reverse transcriptase (Invitrogen, USA) was added to carry out reverse transcription for 2 hr at 50 µl of reaction volume and 42 ° C of reaction temperature, thereby obtaining CyD and Cy5 labeled cDNA. And indirect markers were used ^{3DNA TM Submicro ® EX Expression Array Detection} Kit were compared between the two labeling method.

Labeled cDNAs were purified with MinElute ^™ PCR purification kit (Qiagen, USA), vacuum dried and suspended in 15 μl of TE buffer (10 mM Tris-Cl, pH 8.5) and 8.8 μl of hybridization buffer (10 % SDS (sodium dodecyl sulfate), 20X SSC (sodium chloride / sodium citrate) was added and reacted at 99 ° C. for 5 minutes, cooled at room temperature for 5 minutes, and then introduced into a Hansenula polymorphomicroarray slide at 45 ° C. Hybridization reactions were performed for 16-18 hours.

Hybridized slides were washed sequentially with 2X SSC / 0.2% SDS buffer, 2X SSC buffer, 0.2X SSC buffer, and distilled water and dried by centrifugation at 1,000 rpm.

Slides developed from hybridization reactions were obtained by scanning with ScanArray 5000 (Gsi Lumonics, Kanata, OT, USA) and analyzed using GenePix ^™ Pro 4.0 (Axon Instruments, Foster City, CA, USA) and S-PLUS program. The results were normalized (Stare et al. Computer Methods Programs Biomed. 64, 45-62 (2001)), and the results showed that the expression of the gene was changed more than two times according to the culture conditions. Data clustering was performed using the Cluster program (Eisen et al. Proc. Natl. Acad. Sci. 95, 14863-14868 (1998)) and the TreeView program (http://rana.lbl.gov. EisenSoftware.htm) was used to evaluate schematically.

Table 1 shows the culture of wild-type Hanshenula polymorpha from YPD-2 complex liquid medium to the early stage of exponential growth. The cells are recovered and washed, and transferred to YPM-1 complex liquid medium. Part of the DNA microarray results of Nula polymorpha (Oh et al. J. Microbiol. Biotechnol. 14, 1239-1248 (2004)). As shown in Table 1, the expression rate of methanol oxidase by methanol induction was increased by about 19 times, and the expression rate of catalase was also increased by about 13 times. In addition, catalase showed much higher gene expression than methanol oxidase under oxidative stress. Therefore, based on these results, catalase promoters were selected as methanol-induced promoters related to methanol metabolism.

 Methanol metabolism related enzyme Induction fold Methanol Menadion Methanol oxidase 18.68 One Catalase 12.48 25

Example 2: Acquisition of Hansenula polymorpha CAT promoter

In order to obtain the CAT promoter gene from the above results, Hanshenula from the recently completed genome sequence information of Hanshenula polymorpha RB11 strain (Ramezani-Rad et al. FEMS Yeast Res. 4, 207-215 (2003)). The complete sequence information of the polymorpha catalase ORF (open reading frame) was obtained. On the basis of this, PCR was carried out using a chromosomal DNA of Hanshenula polymorpha A16 strain as a template using primers of SEQ ID NO: 2 and SEQ ID NO: 3 prepared for the top 1,000 to 2,000 bp of the structural gene of Hanshenula polymorpha catalase. Then, a DNA fragment of 1,252 bp containing a Hanshenula polymorpha catalase promoter was obtained (SEQ ID NO: 1). The gene sequence of the Hanshenula polymorpha catalase promoter obtained in the present invention was listed in GeneBank as AY748852, and the gene was cloned into Escherichia coli and deposited with KTCT at KTCC10722BP (Deposit date: November 15, 2004). Was deposited.

Example 3: Verification of Hanshenula polymorpha CAT promoter expression using glucose oxidase reporter gene

In order to compare the expression patterns of the MOX promoter and the CAT promoter by using glucose oxidase (GOD) derived from Aspergillus niger as a reporter protein, a plasmid for expressing GOD using a CAT promoter is illustrated in FIG. 1A. It was produced as shown in. Plasmid pHpCATp-GOD was constructed by replacing the MOX promoter with the CAT promoter from plasmid pDLMOX-GOD (H) (Kang et al. Glycobioligy, 14, 243-251 (2004)) constructed to be expressed by the methanol oxidase promoter. In other words, the 6.3 kb plasmid pDLMOX obtained by cleavage of the Hansenula polymorpha CAT promoter DNA fragments prepared with Bgl II and Hind III restriction enzyme recognition sites at the N- and C-terminus with restriction enzymes BamH I and Hind III. Inserted into -GOD (H) to obtain plasmid pHpCATp-GOD.

Transformation was carried out for each of the above plasmids by lithium acetate-DMSO method (Hill et al. Nucleic Acid Res. 19, 5791 (1991)) in Hanshenula polymorpha DL1 (ATCC26012) and A16 (CBS4732). Transformed recombinant strains were subcultured on YNB minimal plate medium (Yeast Nitrogen Base without amino acid 0.67%, glucose 2%, Leu DO supplement (BD Biosciences Clontech, USA) 0.069%) through leucine nutritional selection The stabilization process was performed and transgenic strains expressing GOD were selected and their expression characteristics were examined.

The general experimental method used for the production of expression plasmids and their transformed strains was in accordance with the method of Kang et al. (Kang et al. Biotechnol. Bioengineer. 76, 175-185 (2001)).

The introduced plasmid copy number of the transforming strain was determined by Southern blotting. Southern blotting was performed according to the method of Kang et al. Biotechnol. Bioengineer. 76, 175-185 (2001).

The expression pattern of GOD by CAT promoter was evaluated by measuring GOD activity in plate medium and liquid culture. Activity measurements on plate media are as follows. Selected transformants were YPD-2, YPD-0.05 (Bacto yeast extract 1%, Bacto peptone 2%, glucose 0.05%), and YPM-2 (Bacto yeast extract 1%, Bacto peptone 2%, methanol 2%) Dispense the seed culture solution with a pipette to form a spot of a constant size on the composite flat medium such as, and incubate at 37 ° C. for 24 to 36 hours, and then pour the top agar for activity measurement onto the flat medium in which the colony is formed and perform a glucose oxidase reaction. Was performed. The activity of the expressed GOD was assessed by chromophores appearing on plate media. Species culture was obtained by incubation at 37 ℃ in 20 hours, YPD-2 complex liquid medium. Top agar solution for activity measurement: 1% agar, 0.7% glucose, 1 U / mL peroxidase (horseradish peroxidase, Sigma), 67 mg / L of O-dianosine (Sigma) in 100 mM potassium ginseng buffer (pH 6.0) It was prepared and used. Liquid culture measurement was carried out by the transformation strain was cultured at 37 ℃ in each complex liquid medium and a certain volume was added to the GOD color reaction aqueous solution to perform the enzymatic reaction, and a certain volume of aqueous hydrochloric acid solution was added to terminate the enzyme reaction. Color development was quantified by measuring absorbance at 525 nm using a UV-spectrophotometer (UV-spectrophotometer, Shimadzu, Japan). Aqueous solution for color reaction was prepared in potassium phosphate buffer solution except agar in the activity measurement top agar solution prepared above.

Figure 2 shows the expression characteristics on the plate medium of Hanshenula polymorpha DL1 and A16 transgenic strain expressing recombinant GOD by CAT and MOX promoters. When the cells were cultured using methanol as the carbon source (YPM), the expression of GOD by the CAT promoter showed a result corresponding to the expression by the MOX promoter. Especially, in the case of the Hansenula polymorph A16 strain, the CAT promoter was MOX. It was relatively better than the promoter (FIG. 2A). And when the cells were cultured with glucose as a carbon source (YPD), it can be seen that the expression of GOD by the MOX promoter was suppressed, but in the case of the CAT promoter, GOD expression was induced (FIG. 2B), In addition, it can be seen that the Hansenula polymorpha A16 strain was more repressed by glucose than in the case of DL1 (FIG. 3). The expression of GOD in glucose and methanol complex liquid medium within 6 hours of incubation showed that the CAT promoter expression by methanol was better than that by glucose, but the expression rate in glucose was significantly higher even at the beginning of culture. Glucose inhibition was found to be significantly alleviated.

Example 4 Expression of Recombinant hSA by Hansenula Polymorpha CAT Promoter

Recombinant human serum albumin (hSA) expression was examined using CAT promoters obtained from PCR of SEQ ID NOs: 2 and 3. The plasmid for hSA expression was digested with plasmid pDLMOX-GOD (H) with restriction enzymes BamH I and Hind III to remove the MOX promoter and GOD gene as shown in FIG. 1B, and then Bgl II and Hind at the N-terminus and C-terminus. The pCAT-BEH was obtained by inserting a Hansenula polymorpha CAT promoter DNA fragment prepared to have a III restriction enzyme recognition site. The hSA gene is derived from pYHSA12 (+/-) (Kang et al. Biotechnol. Bioengineer. 76, 175-185 (2001)) hSA (UTR +) — forward, hSA (UTR −) — forward of SEQ ID NOs: 4, 5 and 6 And a restriction enzyme recognition site of EcoR I and Hind III using respective primers of hSA (UTR +/-)-reverse and containing 5-UTR (untranslated region) of albumin (UTR +) or 5-UTR free (UTR-) hSA gene fragments were obtained. Each hSA gene fragment was inserted into plasmid pCAT-BEH obtained by digestion with EcoR I and Hind III to obtain pCAT-hSA (UTR +) EH and pCAT-hSA (UTR-) EH.

For expression characteristics of hSA by CAT promoter, the selected transformed strains were cultured in YPD-2 or YPM-2 complex liquid medium for 72 hours, and the expression and secretion of hSA were examined through SDS-PAGE. et al. Biotechnol. Bioengineer. 76, 175-185 (2001)).

4 shows the results of examining the characteristics of hSA expression by CAT and MOX promoters. In the case of recombinant hSA expression, expression by CAT promoter was higher than expression by MOX promoter as in the case of recombinant GOD expression of Example 3, and the expression rate of Hansenula polymorpha A16 strain was relatively high. Quantitative analysis revealed that the expression rate of recombinant hSA of CAT promoter was at least 2 to 3 times higher. As shown in the figure, it can be seen that the expression patterns of the two promoters are different from each other.In the case of the MOX promoter, the expression rate increases with the incubation time, but in the case of the CAT promoter, the expression rate of the initial culture is relatively high. .

4B is a result of examining the effect of the presence or absence of 5-UTR, which is a non-translational section above the albumin structural gene, on the expression of recombinant protein during hSA expression. Expression of hSA from the MOX promoter in the results of Hur et al. FEMS Yeast Res. 4, 175-184 (2003) and Kang et al. (Kang et al. Biotechnol. Bioengineer. 76, 175-185 (2001)). In the case of 5-UTR, the expression rate was high, but the glyceraldehyde 3-phosphate dehydrogenase promoter was reported to have a high expression rate when 5-UTR was present. For the present invention, CAT promoter was found to be effective in recombinant protein expression in the absence of 5-UTR as in the case of MOX promoter. These results suggest that in the case of promoters involved in methanol metabolism, the absence of 5-UTR of the protein to be expressed may be effective for protein expression, which suggests that certain metabolic protein translational processes may be used between their promoters and structural genes. It is assumed that it affects regularly from the specific factor of.

Example 5: Expression of Recombinant GFP by Hansenula Polymorpha CAT Promoter

To express another recombinant protein, Green Fluorescent Protein (GFP), from the obtained CAT promoter, plasmid pDLMOX-GOD (H) was cleaved with restriction enzyme Hind III to remove the GOD gene as shown in FIG. 1C. In addition, the restriction enzyme Hind III recognition site by PCR using the pUC19 / yEGFP3 plasmid provided from Nok-Hyun Park and Wonja Choi, Yeast, 19, 1057-1066 (2002) with SEQ ID NOs: 7 and 8 Was inserted a GFP DNA fragment was inserted, thereby obtaining a plasmid pDLMOX-yEGFPm for MOX promoter expression. The plasmid pDLCATp-yEGFPm for expressing CAT promoter was a Hansenula polymorpha obtained by treating plasmid pHPCATp-GOD with restriction enzymes Bgl II and Hind III on a vector DNA fragment obtained by cleaving pDLMOX-yEGFPm with restriction enzymes BamH I and Hind III. Obtained by inserting the CAT promoter gene fragment.

The expression characteristics of GFP by CAT promoter were incubated for 72 hours in selected transgenic strains in YPD-2 or YPM-2 complex liquid medium. The expression level of recombinant protein was determined based on the concentration of the cell at a constant cell concentration (Spectrofluorophotometer, RF 5310PC). , Shimadzu, Japan). The excitation absorbance wavelength for fluorescence measurement of GFP was 488 nm and the emission absorbance wavelength was 510 nm. Expression examination using fluorescence microscopy was performed using Carl Zeiss Laser scanning system (LSM 510 META, Carl Zeiss GmbH, Germany). The excitation and emission wavelengths were the same as in the fluorophotometer analysis conditions.

5 shows the results of examining the expression characteristics of recombinant GFP by CAT and MOX promoters. From the observation by fluorescence microscope, the expression difference between the two promoters in the same culture conditions can be easily confirmed, and in the case of A16, the difference was very clear. In particular, the quantitative results of fluorescence absorbance spectroscopy showed that the expression rate of GFP by CAT promoter was 1.5 times higher than that of MOX promoter. As a result of examining the number of introduction copies of the GFP expression cassette by Southern blotting, 3 copies of the MOX promoter and 2 copies of the CAT promoter were shown. When evaluating the copy number together with the expression rate, the total expression rate was determined by the CAT promoter. 2 times higher.

Overall, the expression system of the recombinant protein provided by the CAT promoter provided in the present invention was up to three times higher than the MOX promoter. In addition, the CAT promoter is not only induction of expression by the methanol carbon source, but also excellent expression even in the presence of a low concentration of the carbon carbon source, it can be seen that it is very effective in recombinant protein production in the fermentation process using glucose as a carbon source. In addition, unlike the MOX promoter, the expression pattern of the promoter is stronger at the early induction, and thus, the cell culture time can be shortened, which has the advantage of preventing the degeneration of the expressed protein due to the long-term culture pointed out in the case of the MOX promoter. .

In other words, in the case of degradation caused by protein hydrolysis, it is advantageous to shorten the culture time and reduce the culture residence time of the generated recombinant protein. In the actual industrial process, the recombinant protein production process by MOX promoter can increase the yield of recombinant protein expression. It is pointed out that the disadvantage of requiring a long period of culture to increase, the CAT promoter provided by the present invention as a solution to such cases is evaluated as very effective.

The promoter of the present invention is a powerful promoter induced by methanol, glucose, and glycerol, and when the promoter is used, the genus Hansenul, Pichia, Candida, or Torulopsis, etc. Medically and industrially useful target proteins can be obtained in high yield in a variety of cells, including yeast.

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Claims (6)

A promoter nucleic acid molecule whose expression is highly induced by methanol or low concentration glucose having a nucleotide sequence of -1252 to -1 of SEQ ID NO: 1 or a nucleotide sequence complementary to the sequence. An expression vector comprising the promoter nucleic acid molecule of claim 1. A host cell transformed with the vector of claim 2. The host cell according to claim 3, which is a yeast. The host cell according to claim 4, which is genus Hansenul, Pichia, Candia, or Torulopsis. Transforming the vector comprising the nucleic acid sequence of the promoter nucleic acid molecule of claim 1 and the nucleic acid molecule encoding the protein of interest into the host cell; Inducing high and early expression of the desired protein in the presence of methanol, glucose, glycerol or a combination thereof; And separating and purifying the protein of interest.

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