KR100618563B1 - Method for Producing Recombinant Proteins in Yeast using cellulose-binding domain - Google Patents

Abstract

The present invention provides a recombinant expression vector operably linked to a regulatory sequence in which a DNA construct having a gene encoding a cellulose binding domain of cellulase at the 5'-end of a gene encoding a target protein is operably linked to yeast, And a method for producing the target protein, characterized by culturing the host cell transformed with the recombinant expression vector, and culturing the host cell and separating and purifying the target protein fused with the cellulose binding domain in the culture thereof. It relates to a target protein fused with a cellulose binding domain prepared from the method.

The present invention also provides a method for producing an immobilized protein in which a protein fused with a cellulose binding domain is attached to the cellulose carrier, comprising culturing the host cell and mixing the culture thereof with a cellulose carrier. It relates to an immobilized protein.

Cellulase, cellulose binding domain, yeast, immobilized protein

Description

Method for Producing Recombinant Proteins in Yeast using cellulose-binding domain

Figure 1 shows the cellulose binding domain amino acid sequence of the cellulase derived from Trigoderma harzianum and the nucleotide sequence encoding the same.

Figure 2A is a schematic representation of a recombinant expression vector pYEGA-AM-Lip comprising a gene encoding a lipase corresponding to one embodiment of the present invention, Figure 2B is a gene encoding a lipase corresponding to an embodiment of the present invention The DNA construct, in which the gene encoding the cellulose binding domain at the end of the DNA construct, is a schematic representation of pYEGA-AM-CBDLip, a recombinant expression vector operably linked to a regulatory sequence acting in yeast.

Figure 3 shows the results of Western blot analysis performed in batch culture of yeast transformed with the recombinant expression vector pYEGA-AM-Lip or pYEGA-AM-CBDLip and to compare the amount of lipase produced, the left lane of the figure Yeast transformed with pYEGA-AM-Lip, the right lane corresponds to yeast transformed with pYEGA-AM-CBDLip.

Figure 4 is a fed-batch culture of yeast transformed with the recombinant expression vector pYEGA-AM-CBDLip, and shows the results of measuring the dry weight of the cells, lipase activity and the concentration of glucose at each time (○: drying of the cells) Weight: ■: extracellular lipase activity; ●: glucose concentration).

FIG. 5 shows the results of SDS-PAGE analysis for confirming the fixation of lipase (CBDLip) fused with cellulose binding domain to cellulose carrier (Avicel), where lane M is a standard molecular weight marker; Lane 1 before mixing of the fused lipase (CBDLip) and the cellulose carrier; Lane 2 corresponds after mixing of the fused lipase (CBDLip) and the cellulose carrier.

Figure 6a shows the change in activity according to the temperature of the lipase (CBDLip) fixed to the cellulose carrier, Figure 6b shows the change in activity according to the pH of the lipase (CBDLip) fixed to the cellulose carrier, Figure 6c It shows the thermal stability according to the temperature of the fixed lipase (CBDLip) (●: Lip; ○: CBDLip; ▲: CBDLip immobilized on the Avicel).

The present invention relates to a method for producing a recombinant protein in yeast using a cellulose binding domain. More specifically, the present invention relates to a DNA construct in which a gene encoding a cellulose binding domain of a cellulase at the 5'-end of a gene encoding a protein of interest is operably linked to a regulatory sequence acting in yeast. Recombinant expression vector and yeast host cell transformed with the recombinant expression vector, the target protein comprising the step of culturing the host cell and separating and purifying the target protein fused with the cellulose binding domain in its culture And a target protein fused with a cellulose binding domain prepared from such a method.

Various mononuclear host cells are used for the production of recombinant proteins. These hosts include E. coli. Well-known eukaryotic and prokaryotic hosts such as E. coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera pruperferda (SF9), animal cells such as CHO and mouse cells, COS 1, African green monkey cells such as COS 7, BSC 1, BSC 40 and BMT 10 and tissue cultured human cells and plant cells.

Among these host cells, Saccharomyces cerevisiae was first used for interferon production in 1981 (Hitzeman et al. Nature, 1981, vol. 293, p. 717), a useful foreign protein. It has been widely used in production. Yeasts can use their intracellular expression and secretion systems to secrete target proteins in their active form and detoxify proteins such as N-terminal methionine removal, glycosylation, and disulfide bonds. It has a post-translation modification system. Yeast has also been classified as GRAS cells (Generally Regarded As Safe), which has been recognized for safety in humans by the US FDA.

As such, when foreign protein is produced using yeast, it is safe for the human body, and can be obtained in a form similar to protein expressed in animal or human cells. There is an advantage of obtaining an active foreign protein immediately without a process such as folding (refolding). However, in the production of recombinant proteins in yeast, the productivity varies considerably from protein to protein, and in most cases the yield is relatively low. Therefore, many researchers have developed various types of expression vectors to try to increase the expression and secretion of proteins in yeast.

For example, European Patent No. 303,833 proposes a method for producing a galactokinase-bovine prochymosin fusion protein in yeast using an expression vector comprising a foreign gene and the GAL1 promoter of yeast. have. Korean Patent No. 97-00097 (Also, Lee et al. Biotechnol. Prog. 1999, Vol . 15, p.884) uses the N-terminal 24 residue of interleukin 1β as a secretion enhancer and secretes it. By inserting the gene with the sequence before the foreign gene, a method of significantly increasing the secretion efficiency of hGCSF and hGH is proposed. European Patent No. 302,723 discloses human interferon-gamma, human serum albumin, tissue plasminogen activator using a promoter and secretory sequence of the alpha-mating factor of yeast. , Rennin, human insulin-like growth factor, and the like are presented.

The present inventors also studied to improve the expression and secretion of recombinant proteins in yeast, when the gene encoding the cellulose binding domain was found to improve the expression and secretion of the recombinant protein when combined with heterologous genes.

Thus, in one aspect, the present invention provides a recombinant recombinantly operably linked to a regulatory sequence in yeast wherein a DNA construct having a gene encoding the cellulose binding domain of cellulase at the 5′-end of a gene encoding a protein of interest is operably linked. Provide an expression vector.

In another aspect, the present invention provides a yeast host cell transformed with the recombinant expression vector.

In another aspect, the present invention provides a method for producing the target protein, comprising culturing the host cell and separating and purifying the target protein fused with a cellulose binding domain in the culture thereof.

In another aspect, the present invention provides a protein of interest fused with a cellulose binding domain.

In still another aspect, the present invention provides a method for preparing an immobilized protein in which a protein fused with a cellulose binding domain is attached to the cellulose carrier, comprising culturing the host cell and mixing the culture thereof with a cellulose carrier. .

In another aspect, the present invention provides an immobilized protein of a protein fused with a cellulose binding domain attached to a cellulose carrier.

In the present invention, when the target protein is produced in a yeast by a genetic engineering method, expression and secretion of the protein are linked to a terminal of a gene encoding the target protein, and preferably a 5'-terminal cellulose binding domain. It's about promoting. Accordingly, the present invention provides a recombinant expression vector operably linked to a regulatory sequence in which a DNA construct having a gene encoding the cellulose binding domain of cellulase at the 5′-end of a gene encoding a protein of interest is operably linked to yeast. Include. When the target protein is produced using the host cell transformed with the recombinant expression vector, the target protein will be expressed and secreted in a fused form with the cellulose binding domain.

As used herein, the term "cellulase" refers to an enzyme that breaks down the polysaccharide cellulose into a disaccharide cellobiose. Most cellulase consists of two domains linked by a linker, one of which binds to solid cellulose and is called the Cellulose-Binding Domain (CBD), while the other is substantially It acts as a catalyst to decompose cellulose and is called a catalytic domain.

Such cellulase is included in fungal cells capable of breaking down cellulose, and in the present invention, all of the cellulose binding domains derived from such cellulase can be used. Preferably cellulase derived from the fungus, more preferably cellulase derived from the Trichoderma sp. Fungus. Fungi in the trigodera include T. atroviridae , T. aureoviridae , T. citrinoviridae , and T. croceum . , T. Lau Kum article (T. glaucum), not annuum T. (T. harzianum), T. cuts matum (T. inhamatum), T. Koh ninji (T. koningii), T. norum League (T. lignorum ) And T. parceramosum . In the present invention, it is most preferred to use the cellulose binding domain of the cellulase derived from Trichoderma hagianum (FIG. 1).

The term "target protein (or protein)" used in the specification of the present invention refers to a protein to be produced by the genetic engineering method according to the present invention, without being particularly limited. It will preferably include proteins that are being used for commercial purposes and need to be produced in large quantities. Proteins include, but are not limited to, serum proteins (eg, blood factors including factors VII, VIII, and IX), immunoglobulins, cytokines (eg, interleukins), α-, β-, and γ-interferons, colony stimulating factors Tissue such as G-CSF and GM-CSF, platelet induced growth factor (PDGF), phospholipase-activated protein (PLAP), insulin, tumor necrosis factor (TNF), growth factor (e.g., TCFα or TGFβ) Growth factor and endothelial growth factor), hormones (e.g. follicle-stimulating hormone, thyroid-stimulating hormone, antidiuretic hormone, pigment and parathyroid hormone, progesterone-releasing hormone and derivatives thereof), calcitonin, calcitonin gene Peptides (Calcitonin Gene Related Peptide (CGPR), enkephalin, somatomedin, erythropoietin, hypothalamic secretion factor, prolactin, chronic gonadotropin, tissue plasminogen activator, growth hormone secretion peptide Beads and the like;; (THF thymic humoral factor) (growth hormone releasing peptide GHPR), thymic humoral factor. In addition, the protein of interest of the present invention will include enzymes, and examples include carbohydrate-specific enzymes, proteolytic enzymes, redox enzymes, transferases, hydrolases, lyases, isomerases and ligases. Specific enzymes include, but are not limited to, asparaginase, arginase, arginine deaminase, adenosine deaminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase , Tyrosinase and bilirubin oxidase. Examples of carbohydrate-specific enzymes include glucose oxidase, glucosidase, galactosidase, glucocerebrosidase, glucoronidase, and the like.

One aspect of the invention relates to a recombinant expression vector comprising a lipase as the protein of interest. The lipase is preferably derived from microorganisms. Lipases derived from microorganisms are particularly advantageous because they are used in various fields such as surfactant, paper and food industry, and organic synthesis.

As used herein, the term “expression control sequence” refers to nucleic acid sequences that are essential or beneficial for the expression of a polypeptide of the invention. Each regulatory sequence can be native or foreign to the nucleic acid sequence encoding the polypeptide. Such regulatory sequences include, but are not limited to, secretory sequences, polyadenylation sequences, propeptide sequences, promoters, enhancer or upstream activation sequences, signal peptide sequences, and transcription terminators, and the like. . In the present invention, the regulatory sequence includes at least a promoter, preferably a promoter and a secretory sequence. Other regulatory sequences can optionally be included to further increase the amount of expression of the polypeptides according to the invention.

In addition, the term "regulatory sequence acting in yeast" as used in the specification of the present invention refers not only to the regulatory sequence originally present in yeast, but also to all regulatory sequences capable of maintaining and exerting a function as a regulatory sequence in yeast.

As used herein, the term "promoter" refers to a nucleic acid sequence essential for initiating transcription of a polypeptide of the invention. Examples of promoters acting in yeast include promoters of the yeast alpha-mating system, promoters of yeast triose phosphatase isomerase (TPI), genes involved in the glycolytic process of yeast (glycolytic genes) or promoters of alcohol dehydrogenase (ADH) genes and enzymes involved in yeast galactose metabolism (e.g., galactose kinase (GAL1) and promoters of uridine diphosphoglucose 4-epimerase (GAL10), GADPH (glyceraldehyde-3-phosphate dehydrogenase), MOX (methano oxidase), AOX (alcohol oxidase) promoter and the like.

As used herein, the term “secretory sequence” (motif sequence, leader sequence or signal peptide) refers to an amino acid sequence that directs the expressed protein to be transported out of the cell membrane. Generally, surface proteins or secreted proteins that are transported out of the cell membrane have an N-terminal sequence that is cut off by a signal peptidase in the cell membrane. In the present invention, secretory sequences include, but are not limited to, alpha-factor signal sequence, killer toxin leader signal sequence, invertase signal sequence, alpha -Amylase signal sequence and the like can be used.

As used herein, the term "vector" refers to a DNA molecule as a carrier that can stably transport foreign genes into a host cell. To be a useful vector, it must be able to replicate, enter into a host cell, and be equipped with means to detect its presence.

As used herein, the term “recombinant expression vector” generally refers to a circular DNA molecule formed operably linked to a vector so that a foreign gene can be expressed in a host cell. Recombinant expression vectors can generate several copies and inserted heterologous DNA. As is well known in the art, to raise the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host. Preferably, the expression control sequence and the gene of interest are included in one expression vector containing the bacterial selection marker and the replication start point together. If the expression host is a eukaryotic cell, the expression vector must further comprise an expression marker useful in the eukaryotic expression host cell.

As used herein, the term “operably linked” refers to a state in which a nucleic acid sequence is placed in a functional relationship with another nucleic acid sequence. This may be genes and regulatory sequence (s) linked in such a way as to enable gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s). For example, if a pre-sequence or secretory sequence functions by participating in the secretion of a mature protein, it is operably linked to that protein. If the promoter has regulated the transcription of the coding sequence, it is operably linked to that sequence. If the ribosomal binding site is in a position where translation of the coding sequence is possible, it is operably linked to that sequence. In general, “operably linked” means that the linked DNA sequences are in contact, and in the case of a secretory leader, are in contact and present within the reading frame. However, enhancers do not need to touch.

The recombinant expression vectors of the present invention described above can be prepared using basic recombinant DNA techniques. According to this, the DNA fragments constituting the recombinant expression vector, specifically, the cellulose binding domain, the target protein, the regulatory sequence and the expression vector must be linked, which is performed by ligation at a suitable restriction enzyme site. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used. In forming the desired recombinant expression vector, the DNA fragments are preferably linked in consideration of a specific order and orientation. Specifically, the method for preparing a recombinant expression vector is as follows.

DNA can be cleaved using specific restriction enzymes in an appropriate buffer. Typically, about 0.2-1 μg of plasmid or DNA fragment is used with about 1-2 units of the restriction enzyme in about 20 μl of buffer solution. Appropriate buffers, DNA concentrations, incubation times and temperatures can be specified by the manufacturer of the restriction enzyme. Generally, incubation at 37 ° C. for about 1-2 hours is appropriate, but some enzymes require higher temperatures. After incubation, the enzyme and other impurities are removed by extracting the digestion solution with a mixture of phenol and chloroform and the DNA can be precipitated with ethanol and recovered from the aqueous layer. It would be more desirable to use commercially available purification kits. In this case, the ends of the DNA fragments must be compatible with each other in order for the DNA fragments to be connected to form a functional vector.

Cleaved DNA fragments are sorted and selected by size using electrophoresis. DNA can be electrophoresed through agarose or polyacrylamide matrices. The choice of matrix depends on the size of the DNA fragment to be separated. After electrophoresis, DNA is extracted from the matrix by electroelution, or if low melting agarose is used, the agarose is melted and the DNA is extracted therefrom.

DNA fragments to be linked are added to the solution in the same molar amount. Such solutions typically contain ligase, such as ATP, a ligase buffer, and about 10 units of T4 ligase per 0.5 μg of DNA. In order for the DNA fragment to be linked to the vector, the vector may be cleaved and linearized with a suitable restriction enzyme and then treated with alkaline phosphatase or bovine hydrolases. This phosphatase treatment prevents self-ligation of the vector during the ligation step. Recombinant expression vectors prepared through such a method are used to transform yeast host cells.

Yeast host cells transformed with the recombinant expression vectors described above constitute another aspect of the present invention. Yeast is described by (1) Simon, MI et al. editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, vol. 194, p. 182-7, Academic Press, Inc. New York; (2) Ito, et al. Journal of Bacteriology, 1983, vol. 153, p. 163; (3) Hinnen et al. Proceedings of the National Academy of Sciences USA, 1978, vol. 75, p, 1920 and (4) Clontech Laboratories, Inc, Palo Alto, Calif., USA (Yeastmaker ^TM Yeast Transformation System Kit), etc. Can be transformed. Examples of yeast host cells usable in the present invention include Saccharomyces sp. (Eg S. cerevisiae), Schizosaccharomyces sp., Klyveromyces sp. Pichia sp. (Eg P. pastoris, P. methanolica), Hansenula sp. (Eg H. polymorpha) and the like.

The present invention provides a method for preparing the target protein, which comprises culturing the transformed yeast host cell and separating and purifying the target protein fused with the cellulose binding domain from the culture thereof.

In the present invention, yeast host cells can be cultured in a nutrient medium suitable for the production of polypeptides using known techniques. For example, the cells can be cultured by small or large scale fermentation, shake flask culture in a laboratory or industrial fermenter carried out under conditions that allow the appropriate medium and polypeptide to be expressed and / or isolated. Cultivation takes place in a suitable nutrient medium, including carbon, nitrogen sources and inorganic salts, using known techniques. Suitable media are available from commercial suppliers and may be made, for example, according to the ingredients and proportions thereof described in the catalog of the American Type Culture Collection. Transformed yeast host cells of the invention can be cultured in batch or fed-batch culture.

"Batch culture" refers to one of the culture modes in which all nutrients are added at the beginning of fermentation. In batch fermentation, organisms grow until one of the essential nutrients in the medium is depleted, or until fermentation conditions become undesirable (eg, when the pH is reduced to a value that inhibits microbial growth).

"Federated cultivation" means a culture in which one or more nutrients are continuously fed to the culture immediately after the start of fermentation or after the culture reaches a stage or when the supplied nutrients are depleted from the culture fluid. do. In fed-batch fermentation, for example, pH control is generally used to maintain the desired growth conditions, and the essential nutrients are fed to the culture to prevent the depletion of one or more essential nutrients. Microorganisms will continue to multiply at a growth rate determined by the nutrient supply rate. In general, a single nutrient, a carbon source, is the limiting factor for growth. Other types of nutrient limits may be available. Other examples include limitations by nitrogen sources, limitations by oxygen, limitations by certain nutrients such as vitamins or amino acids (when the microorganism is a nutritional component for the compound), limitations by sulfur and limitations by phosphorus.

From such cultures the polypeptides of the invention can be isolated by methods known in the art. For example, the polypeptide can be separated from the nutrient medium by traditional methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Further polypeptides are commonly known, including chromatography (eg, ion exchange, affinity, hydrophobicity and size exclusion), electrophoresis, fractional solubility (eg, ammonium sulfate precipitation), SDS-PAGE or extraction It can be purified through various methods. In particular, since the polypeptide of the present invention includes the cellulose binding domain separated from the cellulase, it may be easily separated and purified through chromatography using a cellulose carrier as a column.

As noted above, the target protein prepared by the preparation method according to the present invention will be in a fused form with a cellulose binding domain. The present invention provides the protein of interest in the fused form thus produced, and may itself be used as a final product. In addition, the fused form of the target protein can be used to cut the cellulose binding domain using an appropriate enzyme, etc., and then to produce and use the non-fused form of the target protein through an additional separation and purification process.

The present invention further comprises the step of culturing the yeast transformed with the recombinant expression vector according to the invention and mixing the culture thereof with the cellulose carrier, the immobilized protein attached to the cellulose carrier is fused to the cellulose carrier It provides a method for producing a protein. In the present invention, there is preferably provided a method for preparing an immobilized enzyme, wherein the protein is an enzyme.

The present invention provides an immobilized protein of a protein fused with a cellulose binding domain attached to a cellulose carrier prepared by such a method. In the present invention, there is preferably provided an immobilized enzyme, wherein the protein is an enzyme.

As used herein, "immobilized enzyme" refers to limiting the mobility of an enzyme in a confined space. If the enzyme is immobilized, there are advantages such as enzyme reuse, omission of the recovery and purification of the enzyme, and better conditions may be provided for the enzyme activity. In particular, because enzymes are expensive, reuse of enzymes is a key factor in many processes. Since some of the intracellular enzymes are associated with the membrane, the immobilized enzyme is a model for mimicking and understanding the action of such intracellular enzymes. Another advantage is that the purity of the product is improved, and effluent treatment and problems are minimized.

In preparing such an immobilized enzyme, the protein prepared according to the present invention, specifically, the enzyme is in a fused form with the cellulose binding domain, so if the cellulose carrier is selected as a carrier, the immobilized enzyme can be easily prepared without additional attachment process. will be.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

<Example 1>

Preparation of DNA Sequences Encoding Cellulose Binding Domains

The cellulose binding domain isolates the genomic DNA of Trichoderma hazianum KCTC 16977, which is distributed from the Korea Institute of Biotechnology Gene Bank, and then uses the separated genomic DNA as a template to encode a known cellulose binding domain. Oligonucleotides having the base sequence complementary to the ends of the sequence (SEQ ID NO: 1 and SEQ ID NO: 2; All were included as primers using a restriction enzyme Nde I recognition site) to amplify the gene encoding the cellulose binding domain.

At this time, PCR was mixed with 20pmol primer, 2.5mM dNTP mixture, 100ng plasmid DNA, 5μl 10 x PCR buffer (Solgent Co.), 2.5 unit Taq DNA polymerase so that the final volume is 50μl and then The mixture was denatured at 94 ° C. for 10 minutes, denatured at 94 ° C. for 1 minute, recombined at 55 ° C. for 45 seconds, and stretched for 34 seconds at 72 ° C. for 34 times. The reaction was terminated after expanding for 10 minutes at 72 ° C. The amplified PCR product was electrophoresed on 0.8% agarose gel [agarose gel (BMA, USA)], and then separated using a Qiaex II gel extraction kit (Qiagen, USA).

Example 2

Preparation of pYEGA-AM-Lip

(1) Preparation of pYEGA-AM

As a secretion sequence, the amylase secretion sequence of Aspergillus oryzae was selected for efficient secretion of the desired protein in yeast. DNA encoding a known amylase secretion sequence (SEQ ID NO: 3 and SEQ ID NO: 4: Both restriction enzymes EcoR I and Nde I cleaved site) were synthesized by Genotech.

PYEAG (Ahn et al. J. Microbiol. Biotech. 2003. 13 (3). 451-456), an expression vector acting in yeast, was treated with the restriction enzymes EcoR I and Nde I and then using the CureX Two Gel Extraction Kit. Separated. Subsequently, the DNA encoding the previously synthesized amylase secretion sequence and the expression vector pYEAG were combined with a ligase. The generated recombinant expression vector was named pYEAG-AM.

(2) Preparation of Lipase

Lipase of Bacillus stearothermophilus L1 was selected as the target protein. The lipase The genomic DNA is isolated from Bacillus Steyr Summerphilus, and then the isolated genomic DNA is used as a template, an oligonucleotide having a sequence complementary to the end of a gene encoding a known lipase (SEQ ID NO: 4 and SEQ ID NO: 5). : PCR was performed using primers containing restriction enzymes Nde I and HindII cleavage sites, respectively, to amplify the lipase-encoding gene. At this time, PCR was performed under the same conditions as described in Example 1.

(3) Preparation of pYEG-AM-Lip

The PCR product was electrophoresed on a 0.8% agarose gel [agarose gel (BMA, USA)] and then separated using a Qiaex II gel extraction kit (Qiagen, USA). The PCR product thus separated and each of the expression vector pYEAG-AM (stratagene) prepared in (1) of Example 2 were treated with restriction enzymes Nde I and Hind III, and then separated using CureX Two Gel Extraction Kit. Subsequently, the PCR product treated with the restriction enzyme and the expression vector pYEAG-AM were bound with ligase. The generated recombinant expression vector was named pYEAG-AM-Lip.

Example 3

Preparation of pYEG-AM-CBDLip

Each of the PCR product prepared in Example 1 and the expression vector pYEAG-AM-Lip prepared in (3) of Example 2 was treated with restriction enzyme NdeI. Then, the PCR product treated with the restriction enzyme and the expression vector pYEAG-AM-Lip were linked with ligase. The generated recombinant expression vector was named pYEAG-AM-CBDLip.

<Example 4>

Preparation of Transformant

PYEG-AM-Lip and pYEG-AM-CBDLip prepared in Examples 2 and 3 described above were introduced into Saccharomyces cerevisiae 2805, respectively. In addition, the expression vector was transformed into Saccharomyces cerevisiae yeast, and the resulting yeast was named Saccharomyces cerevisiae / pYEGA-AM-CBDLip, and was deposited with KCTC 10504BP on July 31, 2003 to KCTC. Deposited.

Example 5

Comparison of lipase expression levels of pYEG-AM-Lip and pYEG-AM-CBDLip

The transformants were subjected to batch culture using a medium containing 3% galactose, 1% yeast extract, and 2% peptone, and the expression levels of lipase were compared in the cultures thus formed. The results are shown in Table 1.

As shown in Table 1, lipase expression in the form of a fusion protein linked with cellulose binding domains increased about 7 times more than lipase expressed alone, and the secretion rate was higher.

Comparison of Lipase Expression in Batch Culture of Yeast Transformed with pYEGA-AM-Lip and pYEGA-AM-CBDLip, respectively Expression vector Cell concentration, g / l Lipase activity in culture (U / ml) Intracellular Lipase Activity (U / ml) Secretion efficiency ¹ pYEGA-AM-Lip 15.3 14 11 56 pYEGA-AM-CBD -Lip 15.5 100 21 83

¹ Lipase activity ratio in medium to total expressed lipase activity

In addition, the expressed lipase was confirmed by Western blot (Western blot) method, the results are shown in FIG. According to FIG. 3, the lipase fused with the cellulose binding domain showed a higher molecular weight (61 kDa) than the lipase which was not, and it was found that a large amount was produced.

<Example 6>

Fed-Batch Culture of Yeast Transformed with pYEGA-AM-CBDLip

In order to produce a large amount of lipase, yeast transformed with pYEGA-AM-CBDLip prepared in Example 5 (Accession No. KCTC 10504BP) was subjected to fed-batch culture. Table 2 shows the medium composition used in the fed-batch culture.

Production medium composition used for fed-batch cultivation division Initial Badge ¹ Supply medium ¹ glucose 20 200 Galactose - 200 MgSO ₄ 0.5 5.7 Ammonium Sulfate 5 20 K ₂ HPO ₄ 3 15 CaCl ₂ One One Yeast Extract 5 25 Casein peptone 5 20 Small amount of metal, 10X ² 1ml 2 ml

Composition per ¹ L

² 10x Small amount of metal (NH ₄ ) ₆ MoO ₂₃ 3.0 g, H ₃ BO ₃ 400 g, CuSO ₄ 10 g, MnCl ₂ 80 g, ZnSO ₄ 10 g

First, the transformed yeast was cultured in an initial medium of pH 5.5, temperature 30 ^° C., and when all of the initial glucose 20g / L was consumed, it began to supply the addition medium. The feed rate was increased stepwise from 6 ml / h to 70 ml / h as cell growth was maintained such that the concentration of glucose in the culture was maintained below 1 g / l and the non-cell rate was maintained at 0.03 to 0.05 / h.

At this time, the dry weight lipase activity and glucose concentration of the cells were measured for each fed-batch incubation time. Each measurement method was performed as follows.

(1) Measurement of dry cell amount: The cells were obtained by centrifuging the culture medium, washed with isotonic solution, and dried at 80 ° C. to determine the weight of the cells.

(2) Assay of lipase: The titer of lipase was measured using pH-stat method.

(3) Glucose Assay: Glucose quantification was performed using a glucose analyzer.

The measurement results are shown in FIG. 4, whereby the cells reached a dry cell weight of 95 g / L at 72 hours after incubation, and the lipase was expressed and secreted after feeding of an additional medium containing galactose, which is an expression inducing substance. It was confirmed that the increase with the growth of about 2,200 U / ml at the end of the culture (about 1.2g / L in the amount of protein) was increased.

<Example 7>

Preparation of Immobilized Enzyme by Binding of Lipase to Cellulose Carrier

Cellulose binding domains have been reported in the literature to specifically bind cellulose (Linder et al. Journal of Biotechnology, vol. 57, p. 15). Accordingly, the present inventors attempted to prepare an immobilized enzyme by binding a lipase (CBDLip) fused with the cellulose binding domain prepared above to a cellulose carrier.

Specifically, the fed-batch culture solution described above was separated by centrifugation, and the enzyme solution containing the cells and lipase (CBDLip) was separated and 0.4 mg of cellulose (Avicel) per unit of enzyme was added for about 1 hour. After mixing, the supernatant and lipase (CBDLip) -bound Avicel were separated by centrifugation. It was confirmed through the SDS-PAGE experiment that the lipase (CBDLip) fused with the cellulose binding domain in this process can be effectively bound to the cellulose carrier, and the results are shown in FIG. 5.

According to Figure 5, it can be seen that the lipase (CBDLip) that was present in the enzyme solution is specifically removed after mixing with Avicel, because the lipase (CBDLip) is combined with the cellulose carrier and precipitated together with the carrier through centrifugation.

In order to confirm the activity of the lipase immobilized on the cellulose carrier, the activity change according to temperature, the activity change according to the pH change, and the thermal stability according to the temperature were measured and the results are shown in FIG. 6.

In FIG. 6, the lipase immobilized on the cellulose carrier showed the same enzymatic properties as the lipase which was not. Therefore, it can be seen that the lipase (CBDLip) fused with the cellulose binding domain is effectively immobilized on the cellulose carrier.

Comparative Example 1

Use of other secretion sequences (invertase secretion sequences)

A recombinant expression vector was prepared according to the same procedure as described in Examples 2 to 5 using an invertase signal peptide specified in SEQ ID NO: 6 in place of the amylase secretion sequence used as the secretion sequence in the above-described example. After the yeast transformed with the vector was prepared and cultured, the lipase was isolated and purified from the culture thereof, and the expression level was analyzed. I could confirm it.

Comparative Example 2

Use of other promoters (GAPDH promoters)

A recombinant expression vector was prepared according to the same procedure as described in Examples 2 to 5 using a GAPDH promoter in place of the GAL10 promoter used as a promoter in the above-described examples, a yeast transformed with the vector was prepared and then cultured. As a result of separating and purifying the lipase from the culture thereof, the amount of lipase secretion was improved by about three times when the cellulose binding domain was used.

Comparative Example 3

Use of other target proteins (Candida lipase or Brevibacillus cyclodextrin glycosyltransferase)

Instead of the lipase derived from Bacillus Steyr Summerphilus used as the target protein in the above-described examples, lipase B and Brevibacillus derived from Candida antartica , respectively. brevinos ) -derived cyclodextrin glycosyltransferase according to the same procedure as described in Examples 2 to 5 to prepare a recombinant expression vector, to prepare a yeast transformed with the vector and then culture it, Cyclodextrin glycosyltransferase isolated purified from Candida anthrotica lipase or Brevibacillus brebinose from its culture, and analyzed the expression amount. Increased by fold.

By using the recombinant expression vector and yeast according to the present invention to improve the secretion of the protein of interest in yeast, it is possible to mass-produce the target protein. In addition, the immobilized enzyme immobilized on the cellulose carrier can be prepared using the recombinant expression vector and yeast according to the present invention.

<110> Acebiotech Co. Ltd          Korea Research Institute of Bioscience and Biotechnology <120> Method for Producing Recombinant Proteins in Yeast using          cellulose-binding domain <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ggcatatgca gcaaactgtt tgggggc 27 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggcatatgag agctagttgg cggagt 26 <210> 3 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> signal peptide <400> 3 aattcatgat ggtcgcgtgg tggtctctat ttctgtacgg ccttcaggtc gcggcacctg 60 ctttggctca tatgaaaggt aagcttg 87 <210> 4 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> signal peptide <400> 4 tcgacaagct tacctttcat atgagccaaa gcaggtgccg cgacctgaag gccgtacaga 60 aatagagacc accacgcgac catcatg 87 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gaacatatgg catctccacg cgccaatgat 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gcaaagcttt taaggccgca aactcgccag 30 <210> 7 <211> 114 <212> DNA <213> Artificial Sequence <220> <223> signal peptide <400> 7 atgcttttgc aagctttcct tttccttttg gctggttttg cagccaaaat atctgcctac 60 gaaaacgttc gaaaggaaaa ggaaaaccga ccaaaacgtc ggttttatag acgg 114 <210> 8 <211> 210 <212> DNA <213> Artificial Sequence <220> <223> cellulose biding domain <400> 8 cagcaaactg tttgggggca atgcggaggt caaggatgga gcggcccgac tagttgcgtt 60 gctggatctg cttgttctac tctcaacccc tactacgctc aatgcattcc tggagccact 120 accatgtcta ccacaaccaa gccgacctcc gtttcagcat caacgaccag ggcgagtgca 180 acatcgtccg ctactccgcc aactagctct 210 <210> 9 <211> 70 <212> PRT <213> Artificial Sequence <220> 223 cellulose binding domain <400> 9 Gln Gln Thr Val Trp Gly Gln Cys Gly Gly Gln Gly Trp Ser Gly Pro   1 5 10 15 Thr Ser Cys Val Ala Gly Ser Ala Cys Ser Thr Leu Asn Pro Tyr Tyr              20 25 30 Ala Gln Cys Ile Pro Gly Ala Thr Thr Met Met Thr Thr Thr Thr Lys Pro          35 40 45 Thr Ser Val Ser Ala Ser Thr Thr Arg Ala Ser Ala Thr Ser Ser Ala      50 55 60 Thr Pro Pro Thr Ser Ser  65 70

Claims (20)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete Operably linking a gene encoding a protein of interest to a regulatory sequence acting in yeast to produce a recombinant expression vector; (2) transforming the yeast host cell with the recombinant expression vector; And (3) culturing the transformed yeast host cell and separating and purifying the target protein from the culture thereof. A method of enhancing the expression and secretion rate of the target protein by introducing the nucleotide sequence of SEQ ID NO: 8 at the 5'-end of the gene encoding the target protein.

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