KR101802980B1 - Novel translational fusion partner derived from Pichia pastoris for secretory production of foreign proteins and use thereof - Google Patents

Abstract

The present invention relates to a method for producing yeast Pichia pastoris) of the strain-derived target protein secretion production protein fusion factor for, a polynucleotide, a recombinant expression vector, the transformant and the protein fusion factor containing the polynucleotide or recombinant expression vector containing the same coding for the protein fusion factor And a method for producing a target protein.

Description

[0002] Protein fusion factors for producing secreted protein of interest derived from Pichia pastoris strain and uses thereof [0002]

The present invention relates to a protein fusion gene for production of a desired protein derived from a yeast Pichia pastoris strain, a polynucleotide encoding the protein fusion factor, a recombinant expression vector comprising the polynucleotide or the recombinant expression vector And a method for producing a target protein using the protein fusion factor.

It is necessary to develop a high-efficiency protein production system using recombinant microorganisms to analyze the genome sequence information obtained from the human genome project and the functions of various proteins revealed in the genome unit and to produce protein products which are important for human medicine. When selecting an expression system for the production of recombinant proteins derived from higher organisms such as the human body, the growth characteristics of the host cells, the degree of protein expression, the possibility of expression in the cells, the possibility of post-translational modification, The use of active and expressed proteins, and the like.

Escherichia coli and yeast system are mainly used as a representative microorganism gene expression system for producing recombinant protein. Escherichia coli has many expression systems and has a very high expression rate of target protein. However, when recombinant production of proteins derived from higher organisms is desired Glycosylation, and it is difficult to complete the secretion of proteins by the culture medium of the cells, and it is impossible to fold the protein with many disulfide bonds and the inclusion bodies And insoluble protein forms (Makrides, Microbial Rev., 1996, 60, 512).

Pichia pastoris , a eukaryotic microorganism, is the most widely used microorganism for production of recombinant proteins together with Saccharomyces cerevisiae . It is easy to genetically manipulate and various expression systems have been developed and mass culture This is easy. In addition, when recombinant production of proteins derived from high-level cells such as human proteins is performed, proteins can be secreted out of the cells, and the function of post-translational modification of proteins such as sugar chain addition can be performed do. Secretory production of recombinant protein is possible by extracellular secretion by artificially fusing the protein secretion signal and target protein. Protein folding, formation of disulfide bond, and sugar chain addition process are progressed through the secretion process of protein, Lt; RTI ID = 0.0 > recombinant < / RTI > protein. This is also economical because it does not require the economically inefficient cell grinding or refolding steps because proteins with biological activity can be obtained directly from the medium (Eckart and Bussineau, Curr. Opin. Biotechnol., 1996, 7, 525).

Although there have been studies for secreting expression of recombinant proteins in Pichia pastoris strains for a long time, to date there are only a few signal peptides applicable to the production of recombinant proteins. Among them, the signal peptide of MFα (mating factor alpha) derived from Saccharomyces cerevisiae, a yeast system which is mainly used, is the most widely used strong signal peptide in Pichia pastoris and Saccharomyces strains. However, despite the ability to induce the secretory expression of MFα signal peptide, the same effect can not be expected in all proteins. In the case that secretion expression can not be achieved even using MFα signal peptide, there is no suitable alternative.

Under these circumstances, the inventors of the present invention have conducted intensive research to develop a method for producing various target proteins more effectively, and as a result, it has been found that protein fusion factors for producing secreted protein of interest derived from yeast Pichia pastoris strain are selected And confirming that the expression of interleukin-2, which is a type of micronutrient protein, is increased, thereby confirming that the target protein can be secreted more easily, thus completing the present invention.

One object of the present invention is to provide a method for producing a desired protein from a yeast Pichia pastoris strain comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 13, (TFP, translational fusion partner).

Another object of the present invention is to provide a fusion protein having increased secretion ability in yeast, comprising the fusion protein for protein production and the protein of interest.

Yet another object of the present invention is to provide a polynucleotide encoding said protein fusion factor.

It is still another object of the present invention to provide an expression vector comprising the polynucleotide.

It is still another object of the present invention to provide a transformant comprising the expression vector.

It is still another object of the present invention to provide a method for producing a target protein, which comprises culturing yeast into which the expression vector has been introduced.

In order to achieve the above object, one embodiment of the present invention provides a newly isolated protein fusion factor. Specifically, the present invention relates to a protein fusion factor that secretes and produces a target protein in yeast, comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 13 or fragments thereof. Specifically, the yeast is Saccharomyces cerevisiae S. cerevisiae , or Pichia pastoris .

The yeast of the present invention, " Pichia & Pastoris "is a microorganism most commonly used for the production of recombinant proteins together with Saccharomyces cerevisiae . It is easy to genetically manipulate, and various expression systems have been developed and mass culture is easy. In addition, (Eg, secretory proteins that secrete proteins outside the cell when recombinantly produced high-level cell-derived proteins), and post-translational modification functions of proteins such as sugar chain addition. The secretion of the recombinant protein is produced by artificially fusing the protein secretion signal and the target protein to produce an extracellular secretion. Protein secretion through the process of protein folding Or disulfide bond, and sugar chain addition process, and thus it is possible to produce a recombinant protein having a biologically complete activity. Further, since a protein having biological activity can be directly obtained from a medium, However, a suitable protein secretion signal capable of expressing a target protein with a high secretion function in a yeast used as a production host for such a protein drug such as Pichia pastoris, etc., There were shortcomings.

Thus, the present inventors confirmed that a newly isolated protein fusion protein derived from Pichia pastoris secretes interleukin-2, which is a representative embryogenic protein, with high efficiency, and has reached a point of providing a novel protein fusion factor.

The term " translational fusion partner (TFP) " of the present invention refers to a gene that fuses with a gene encoding a hair-disordered protein in a recombinant yeast expression system to induce the secretory production of a hairless protein, 1, 3, 5, and 13, or a fragment thereof. However, it is not limited thereto, and it may be one or more amino acid sequences selected from the group consisting of the above-mentioned sequences or fragments Mutants may also be included.

Specifically, yeast Pichia pastoris) as a strain derived can be exemplified pTFP-1 (pichia translational fusion partner -1) to pTFP-8, etc. (Table 2). As shown in FIG. 5, in the case of transformants into which pTFP1-hIL2, pTFP2-hIL2, pTFP3-hIL2, pTFP4-hIL2 or pTFP8-hIL2, among pTFP-1 to pTFP-8, HIL-2 constructed in seven kinds of pPINK-LC vectors of Invitrogen was hardly expressed, whereas hIL-2 was secreted by using MFa signal peptide derived from Saccharomyces cerevisiae introduced into Pichia In the case of the transformants expressing the transformants and the transformants into which pTFP1-hIL2, pTFP2-hIL2, pTFP3-hIL2, pTFP4-hIL2 or pTFP8-hIL2 were introduced, it was confirmed that the expression of the interspecific protein interferon-2.

The term "target protein " of the present invention means a protein to be produced in a host cell. The present invention refers to a protein which is difficult to produce recombinant expression in a host cell such as Escherichia coli or yeast due to the characteristics of the protein itself when recombinantly producing proteins derived from human or various living organisms. In addition, although recombinant production is possible in the host cell, yeast may include a large number of proteins having low productivity due to low productivity, but may include any protein to be produced in a host cell without limitation.

Specifically, the target proteins include, but are not limited to, serum proteins (blood factors including Factors VII, VIII and IX), immunoglobulins, cytokines (interleukins), alpha-, (TNF), growth factors (e.g., TGF-β), growth factors (eg, GM-CSF), epithelial growth factor (EGF), platelet derived growth factor (PDGF), phospholipase- hormone (thyroid-stimulating hormone, antidiuretic hormone, pigmentary hormone and parathyroid hormone, luteinizing hormone and its analogues), calcitonin ), Calcitonin gene related peptide (CGPR), enkephalin, somatomedin, erythropoietin, hypothalamic secretory factor, prolactin, chronic gonadotropin, tissue plasminogen activator, growth hormone secretion Peptide, and the like;; (THF thymic humoral factor) (growth hormone releasing peptide GHPR), thymic humoral factor. Such proteins will also include enzymes, for example, carbohydrate-specific enzymes, proteolytic enzymes, redox enzymes, transferases, hydrolases, lyases, isomerases and ligases. But are not limited to, asparaginase, arginine, arginine deaminase, adenosine deaminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, ukase, adenosine diphosphatase, tyrosinase and bilirubin Oxydase. Examples of carbohydrate-specific enzymes include glucose oxidase, glucosidase, galactosidase, glucocerebrosidase, glucuronidase, and the like. More specifically, it may be interleukin-2.

Another embodiment of the present invention relates to a separated fusion protein having increased secretion ability in yeast, comprising the protein fusion protein and the target protein for producing the desired protein secretion. The protein fusion factor and the target protein for production of the target protein may be directly connected to each other through a peptide bond or a disulfide bond, or may be connected to each other through a linker.

In the present invention, the term "linker" basically refers to two different fusion partners (for example, biological polymers, etc.) with hydrogen bonding, electrostatic interactions, Van der Waals forces, disulfide bonds, Hydrophobic interaction, covalent bonding, or the like. Can have at least one cysteine capable of participating in at least one disulfide bond, preferably under physiological conditions or other standard peptide conditions (e. G., Peptide purification conditions, peptide storage conditions) In addition to the role of hinge, it is also possible to perform a role of providing a gap of a certain size between the fusion partners or as a hinge which provides flexibility or rigidity to the combination. In addition, it includes a site that can be separated by an in vivo enzyme or the like, and can facilitate separation and purification of the target protein. The linker may be a non-peptide linker or a peptide linker, but is not limited thereto.

In one embodiment of the present invention, a human interleukin-2 protein, which is a type of micronization active protein, is ligated with pTFP as the target protein to transform yeast Saccharomyces cerevisiae and Pichia pastoris strain, (Fig. 4 to Fig. 6). In particular, it was confirmed that the expression of the protein in the Pichia pastoris was remarkably increased.

Another embodiment of the present invention may be a polynucleotide encoding the protein fusion factor. Specifically, the polynucleotide may be a polynucleotide having one species selected from the group consisting of SEQ ID NOS: 2, 6, 8 and 14 Or more of the nucleotide sequence. The polynucleotide may be DNA or RNA, and when the polynucleotide of the present invention is RNA, it can be understood that T (thymine) of DNA is replaced with uracil (U). The polynucleotide can be produced by a known chemical synthesis method.

Another embodiment of the present invention may be an expression vector comprising the polynucleotide. Specifically, the expression vector may include a nucleic acid encoding a target protein.

The term " expression vector " as used herein means a gene capable of expressing a target protein or a target RNA in a suitable host cell, and a gene construct comprising an essential regulatory element operatively linked to the expression of the gene insert (the polynucleotide) . The expression vector can be replicated independently of the host chromosomal DNA once in the host cell, and the inserted foreign DNA can be expressed.

The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoter, operator, initiation codon, termination codon, polyadenylation signal and enhancer, and can be prepared variously according to the purpose. The promoter of the vector may be constitutive or inducible. The expression vector may also include a selection marker for selecting a host cell containing the vector and, if the expression vector is a replicable vector, a replication origin. In particular, in the present invention, it was confirmed that the expression amount of the micronutrient protein was increased by using the pTFP factor as a signal sequence. As a result, the expression vector of the present invention was found to be a yeast Pichia and a protein fusion factor for production of a target protein secreted from a strain of P. pastoris .

Another embodiment of the present invention relates to a transformant comprising the polynucleotide or a recombinant vector comprising the polynucleotide.

The term "transformation" of the present invention means that DNA is introduced into a host and the DNA becomes replicable as an extrachromosomal element or by chromosome integration completion. The host cell that can be used for transformation according to the present invention may include both prokaryotic or eukaryotic cells, and a host having high efficiency of introduction of DNA and high efficiency of expression of the introduced DNA may be used. For example, known eukaryotic and prokaryotic hosts such as Escherichia, Pseudomonas, Bacillus, Streptomyces, Fungi, Yeast, insect cells such as Spodoptera prougiperata (SF9), CHO, COS 1, COS 7, BSC 1, BSC 40, and BMT 10, and the like, but the present invention is not limited thereto. More specifically, for the purpose of the present invention, in order to produce a fusion protein in which the target protein is fused, it may be a genus of Pichia and Saccharomyces, and a genome of Pichia pastoris pastoris ) or yeast including Saccharomyces cerevisiae .

Transformation includes any method of introducing a polynucleotide, and can be carried out by selecting a suitable standard technique depending on the host cell as is known in the art. Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, agrobacteria-mediated transformation, PEG (polyethylene glycol) Dextran sulfate, lipofectamine, particle bombardment, and the like.

In one embodiment of the present invention, when the transformant was transformed into Pichia pastoris , the hIL-2 protein expression of pTFP1-hIL2 was 150% higher than that of the control MFα-hIL2 , And the protein expression of pTFP4-hIL2 was 280% higher than that of the control MFα-hIL2 (FIG. 6).

The present inventors have also found that transformants prepared by introducing pGAPZ / pTFP1-hIL2, pGAPZ / pTFP2-hIL2, pGAPZ / pTFP3-hIL2 and pGAPZ / pTFP8-hIL2 into Escherichia coli DH5α, respectively, KCTC18302P, KCTC18303P, and KCTC18305P were deposited with the Korean Collection for Type Culture (KCTC) on June 17, 2014, and deposited with the International Depository under the Budapest Treaty on June 9, 2015 And received deposit numbers KCTC12833BP, KCTC12834BP, KCTC12835BP, and KCTC12837BP, respectively. Accordingly, the transformant of the present invention may be, but is not limited to, those of deposit numbers KCTC18301P (KCTC12833BP), KCTC18302P (KCTC12834BP), KCTC18303P (KCTC12835BP) or KCTC18305P (KCTC12837BP). The transformant having the E. coli as a host can be used for the proliferation of a protein fusion factor or a vector containing the protein fusion factor.

Another embodiment of the present invention relates to a method for producing a target protein, comprising culturing a yeast into which the expression vector has been introduced in a medium. Specifically, the yeast may be Pichia pastoris or Saccharomyces cerevisiae. However, the host capable of increasing the secretion and / or expression amount of the target protein by the protein fusion factor of the present invention may be included And the target protein may be Interleukin-2. In addition, in the present invention, the production of the target protein may be increased in the secretory ability as compared with the transformant not containing the protein fusion factor, so that mass production is possible.

Specifically, the transformant may be cultured in a culture medium containing a carbon source and a nitrogen source necessary for culturing a conventional yeast or a strain. In addition, a non-ionic surfactant, Polysorbate (trade name tween20) Or a medium comprising poloxamer (trade name Poloxamer 188).

In more detail, the method of the present invention may further comprise the step of recovering the target protein.

The method for preparing a target protein of the present invention may further comprise the step of purifying the recovered target protein, and purification of the target protein may be performed by immunoaffinity chromatography, receptor affinity chromatography, hydrophobic effect chromatography, lectin Can be carried out by conventional chromatographic methods including affinity chromatography, size exclusion chromatography, cation or anion exchange chromatography, high performance liquid chromatography (HPLC) and reverse phase HPLC, and the like. Further, there is a method in which a desired protein is a fusion protein having a specific tag, label, or chelate moiety so that it can be recognized and purified by a specific binding partner or agent. The purified protein may be truncated to the desired protein portion or may remain as such. Cleavage of the fusion protein can result in the desired protein form with additional amino acids in the cleavage process.

In the present invention, "oil-in-water culture" is a culture method in which medium is intermittently supplied, and the concentration of the substrate in the culture medium can be arbitrarily controlled. Since the substrate is added at a proper rate and there is no outflow, Which is the most common culture method. The term " culture medium "

In one embodiment of the present invention, the transformed yeast transformed with pTFP4-hIL-2 was cultured in a 5 L fermenter (Example 8), the transformant was grown normally, and interleukin-2 And secreted at a high concentration (Fig. 14).

In another embodiment (Example 9) of the present invention, the recovery rate of the target protein in the case of additionally including the step of adding detergent in the fermentation production and the case of not adding the washing solution were compared, It was confirmed that the recovery of the protein was high (Figs. 15 and 16).

In another embodiment of the present invention (Example 10), the purified hIL-2 protein was added to the EL-4 cell line at different concentrations to confirm lymphocyte proliferation. As a result, hIL-2 protein overexpressed using pTFP4, (Fig. 17). ≪ tb > < TABLE >

By using the protein fusion factor of the present invention, it is possible to produce a large amount of protein which is difficult to mass-produce in yeast by conventional recombinant technology, and to produce recombinant protein using yeast expression system which has not been widely used for recombinant protein expression with low productivity Can be widely used for the production of proteins.

FIG. 1 is a schematic diagram showing a polymerase chain reaction (PCR) and intracellular recombination process for introducing secretory signal genes derived from Pichia pastoris into a vector for screening pTFP using an invertase system.
FIG. 2 is a schematic diagram showing a polymerase chain reaction (PCR) and an intracellular recombination process for introducing a human interleukin-2 gene, which is an immunosuppressive protein, into various sizes of pTFP vectors using an invertase system.
FIG. 3 is a schematic diagram showing a polymerase chain reaction (PCR) and intracellular recombination process for introducing the selected seven pTFP-hIL-2 genes into a YGa expression vector for yeast Saccharomyces cerevisiae.
Fig. 4 is a graph showing the results obtained by comparing YGa / pTFP1-hIL2, YGa / pTFP2-hIL2, YGa / pTFP3-hIL2, YGa / pTFP4-hIL2, YGa / pTFP5- hIL2, YGa / pTFP7- And the culture supernatant of each of the three single transformants introduced and analyzed was analyzed by SDS-PAGE.
PGAPZ / pTFP4-hIL2, pGAPZ / pTFP3-hIL2, pGAPZ / pTFP4-hIL2, pGAPZ / pTFP4-hIL2, And pGAPZ / pTFP8-hIL2 were respectively introduced into the culture supernatant of the transformant by SDS-PAGE.
Fig. 6 is a graph showing the results of the expression of pGAPZ / pTFP1-hIL2, pGAPZ / pTFP1-hIL2, pGAPZ / pTFP2-hIL2, pGAPZ / pTFP3-hIL2, pGAPZ / pTFP4- The degree of expression in Kiapastris was analyzed by SDS-PAGE and Western blotting.
Fig. 7 shows the results of the Southern blot analysis of pGAPZ / pTFP1-hIL2 and pGAPZ / pTFP4-hIL2, which are highly transfected with human interleukin-2 protein, Southern blot hybridization).
8 is a schematic diagram showing the optimal sequence of pTFP1 that maximizes protein expression using pGAPZ / pTFP1-hIL2 and dividing the pTFP1 portion by five characteristics.
9 is a schematic diagram showing a polymerase chain reaction (PCR) and an intracellular recombination process for introduction into a pGAPZ vector in yeast Pichia pastoris.
10 is a schematic diagram showing an expression vector of pGAPZ / pTFP1-hIL2 gene expressible in Pichia pastoris.
11 is a schematic diagram showing an expression vector of pGAPZ / pTFP4-hIL2 gene expressible in Pichia pastoris.
PTFP1-1-hIL2, pGAPZ / pTFP1-2-hIL2, pGAPZ / pTFP1-3-hIL2, pGAPZ / pTFP1-4-hIL2 and pGAPZ / pTFP1-5-hIL2, The supernatant of the culture of hIL2 transformant was analyzed by SDS-PAGE.
Fig. 13 shows the results of analysis of cell growth by measuring cell concentration over time from a medium obtained by fermenting fermentation in a 5 L fermenter with a recombinant vector containing pGAPZ / pTFP4-hIL2 gene and fermenting in a 5 L fermenter will be.
14 shows the expression of human interleukin-2 protein by SDS-PAGE from a medium obtained by fermentation and fermentation in a 5 L fermenter with a recombinant vector containing pGAPZ / pTFP4-hIL2 gene The results are shown.
FIG. 15 shows a chromatogram for purifying hIL protein produced by fermenting a Pichia pastoris strain transformed with a recombinant vector containing the pGAPZ / pTFP4-hIL2 gene.
FIG. 16 shows the results of purifying the hIL protein produced by fermenting a Pichia pastoris strain transformed with a recombinant vector containing the pGAPZ / pTFP4-hIL2 gene.
FIG. 17 shows the results of confirming the activity of the hIL2 protein produced by fermenting a Pichia pastoris strain transformed with a recombinant vector containing the pGAPZ / pTFP4-hIL2 gene.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1: Strain used and experimental material

Pichia pastoris GS115) buy a strain in-Tron Corporation (Invitron, USA), and YPD (1% yeast extract, 2% Bacto peptone, 2% glucose) medium or YPDS (1% yeast extract, 2% Bacto peptone, 1% glucose, 1% sorbitol) at 30 DEG C for 3 days. In addition, Saccharomyces cerevisiae was transformed with YPSGA medium (1% yeast extract, 2% bactopeptone, 2% sucrose, 0.3% galactose, 2 ug / ml antimycin, 17 ug / Ml of chloramphenicol), and the invertase-free transformants were selected from SD Ura-medium (yeast nitrogen base lacking 0.67% amino acid, 2% glucose, 0.5% casamino acid), and YPDG (1% yeast extract , 2% bactopeptone, 1% glucose, 1% galactose) for 40 hours, and 0.6 ml of the supernatant was precipitated with 0.4 ml of acetone, followed by SDS-PAGE analysis. Escherichia coli DH5α was used for general gene manipulation of the selected transformants.

Example 2. Genetic manipulation and nucleotide sequence analysis method

General gene manipulation was performed according to techniques such as Sambrook (Molecular cloning: a laboratory manual, 2nd ed, 1989) and a gel extraction kit (Viogene) was used to recover the genes from the agarose gel. Genomic DNA was extracted from Pichia pastoris GS115 according to the method used by Connie Holm and Douglas W. Meeks-wagnerd (Gene. 42, 169-173, 1986) and Gietz (Yeast 11, 355-360 , 1995), and the like. E. coli was transformed using Inoue (Gene 96, 23-28, 1990). The nucleotide sequence of the gene was analyzed using Model 373A from Applied Biosystems. Primers to be used for polymerase chain reaction were synthesized by Genotech.

Example  3. Pikia Pastor  Derived For protein secretion factor (pTFP)  Library production

Pichia pastoris GS115 ( Pichia In order to construct a protein fusion gene library from genomic DNA, 69 proteins known to be secreted outside the cell or secreted in the cell membrane of the secretory protein sequence of P. pastoris were selected and primer sequences for amplifying each gene were selected Respectively.

The Pichia pastoris genomic DNA was recovered by the method described in Examples 1 and 2, and a sense / antisense primer corresponding to each gene sequence was prepared so as to be able to secure about 69 kb of the genomic DNA including the secretory signal sequence of about 1 kb, PCR was carried out using a pair of primers (94 ° C for 4 minutes, 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute, 25 times at 72 ° C, Once every 7 minutes). Then, each amplified gene was recovered through agarose gel electrophoresis. The recovered polymerase chain reaction products were quantified using nano drop (thermo scientific, USA) and mixed at the same concentration. Each gene amplified by this method has the same 5 'sequence required for unidirectional PCR.

The unidirectional polymerase chain reaction was performed using only the sense primer (GalSfiI of SEQ ID NO: 15) as a template (100 ng of the above mixed product) (reaction at 95 ° C for 20 seconds, 65 ° C for 10 seconds, 72 ° C for 30 seconds, ) To amplify only a single strand of the gene. From this, a double-stranded gene of various sizes was prepared. The PCR product was subjected to reaction at 37 ° C using a random primer (SfiB3'N6 of SEQ ID NO: 16) and dNTP and a Klenow fragment (exo) Respectively. After 1 hour of reaction, 0.3 to 0.6 kb of DNA was recovered from the agarose gel. Then, the recovered DNA was amplified once again with a sense GalSfiI (SEQ ID NO: 15) / antisense SfiB3 '(SEQ ID NO: 17) primer to recover DNA of 0.2 to 0.8 kb in agarose gel.

YGaINV vector having yeast and E. coli shuttle vector and having SUC2 maturation sequence (513 amino acids) was digested with Saccharomyces cerevisiae Y2805Δgal1Δsuc2 (Mat a ura3 suc2 :: Tcl90 pep4: : HIS3 gall canl) (Korean Patent No. 975,596). The PCR reaction and the recombination process are schematically shown in FIG. The transformed cells were each plated on YPSGA medium (1% yeast extract, 2% bactopeptone, 2% sucrose, 0.3% galactose, 1 쨉 g / ml antimycin A and 2% agar) and cultured for 5 days.

When the target gene is in-frame-linked via intracellular recombination into a vector containing a suitable Pichia Translational Fusion Partner (pTFP) derived from a suitable Pichia pastoris, the invertase having activity is secreted into the medium It is possible to grow on YPSGA medium using sugar as a carbon source. This method was used to preferentially select 1500 optimal pTFPs that induce the secretion of the invertase protein. All transformants were recovered using sterile distilled water, and a fused genomic library plasmid overexpressing the invertase was obtained from the cells using a whole-plasmid extraction kit (Bioneer, Korea). The recovered plasmids were transformed into DH5α and plated on ampicillin-containing 2YP medium (1.6% bactotryptone, 1% yeast extract, 0.5% NaCl, ampicillin 100 μg / ml) 2 x 10 < 6 > cells were obtained, and the sequences of the plasmids randomly selected from the library after DNA plasmid extraction were analyzed. As a result, it was found that all the pTFPs analyzed were derived from Pichia pastoris Gene.

Example 4 Preparation of a New Protein Fusion Factor Overexpressing Interleukin-2

The YGadV45 vector, in which 45 amino acids of the invertase amino terminus were removed by linking mature human interleukin-2 (mature hIL-2) gene, known as a representative egg expression protein, with about 1,500 pTFPs capable of overexpressing the invertase Patent No. 975,596). For this purpose, 1,500 pTFP plasmids were used as a template and polymerase chain reaction (once at 94 DEG C for 5 minutes; at 95 DEG C for 20 seconds, using sense primer Gal100 (SEQ ID NO: 18) and antisense primer LDKR42 (SEQ ID NO: 25 cycles of reaction at 53 DEG C for 30 seconds, 72 DEG C for 30 seconds, and 72 minutes at 72 DEG C for 7 minutes) to obtain 0.2 to 0.8 kb PCR products from the agarose gel. The 5 'end of the PCR product is complementary to the 3' end of the Gal10 promoter of the YGadV45 vector to be introduced. The 3 'end of this product is complementary to the 5' end of the human interleukin-2 gene of the target protein. To amplify the human interleukin-2 gene to be ligated to pTFP, the human interleukin-2 gene used in Korean Patent No. 975,596 was polymerized using the sense primer LNK39 (SEQ ID NO: 20) and the antisense primer CR139 (SEQ ID NO: 21) The 5 'end of the product was inserted into the 3' end of the pTFP by enzyme-linked reaction (once at 94 ° C for 5 minutes, at 94 ° C for 30 seconds, at 55 ° C for 30 seconds, at 72 ° C for 30 seconds, A product of about 440 bp in size with a sequence complementary to the end was obtained. In order to amplify the 45 amino acids at the 5 'end of the insufficient invertase in the YGadV45 vector, the PCR was carried out using the sense primer CR138 (SEQ ID NO: 22) and the antisense primer CR142 (SEQ ID NO: 23) 25 minutes at 72 ° C for 30 minutes, 72 ° C for 7 minutes at once), from which a sequence complementary to the 3 'end of human interleukin-2 A product of about 170 bp in the size of the 5 'terminal sequence was obtained.

The three PCR products obtained were subjected to Sfi I-treated YGadV45 vector and then subjected to three piece in-line recombination with three kinds of fragments in Y2805Δgal1Δsuc2 (Korean Patent No. 975,596) strain (Mat a ura3 suc2 :: Tcl90 pep4 :: HIS3 gall canl) vivo recombination method. The PCR reaction and the recombination process are schematically shown in FIG. The transformed cells were cultured in a Uracil-free selective medium UD (0.67% amino acid free yeast nitrite base, 0.77 g / l amino acid mixture, 2% glucose, 2% agar) medium and YPSGA medium (1% yeast extract, 2% bactopeptone, 2% sucrose, 0.3% galactose, 1 μg / ml antimycin A, 2% agar) and cultured for 5-7 days. From this, about 2 X 10 4 transformants were formed in UD, but about 150 transformants were formed in YPSGA medium, and 15 transformants which formed large colonies by in vitro secretion were selected. The pTFP portion of each transformant was sequenced to confirm the sequence of seven novel human IL-2 over-expressing pTFP regions with different kinds of genes or different amino acid numbers, and are shown in Table 2 below. pTFP1, 4, and 7 were the same gene but they were identified in different sizes. At the 3 'end, there was a linker site of 15 or more amino acids including the Kex2 recognition sequence with hIL-2.

Example  5. Yeast S. cerevisiae  In pTFP - confirmation of expression of interleukin-2

Since the expression vector obtained in the above example is expressed by interleukin-2 and invertase, expression of the interleukin-2 gene is removed and the interleukin-2 gene is expressed. The pTFP-hIL2 portion was amplified by polymerase chain reaction (94 ° C for 5 minutes; 94 ° C for 30 seconds, 55 ° C) with sense primer Gal100 (SEQ ID NO: 18) and antisense primer CR143 30 sec, 72 ° C for 1 min, 25 times at 72 ° C for 7 min), and the first recovered fragments were used as a template to amplify the sense primer Gal100 for introducing a sequence complementary to the YGa vector end without the invertebrate gene (94 ° C for 5 minutes, 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute) using a primer (SEQ ID NO: 18) and an antisense primer GT50R At 25 ° C for 7 minutes at 72 ° C) to prepare a gene capable of in vivo recombination with the vector in yeast. The gene amplified by this method is a structure in which the invertase gene is removed and a protein synthesis termination codon is introduced at the hIL-2 gene end. FIG. 3 shows a polymerase chain reaction (PCR) and intracellular recombination process for introducing the selected seven kinds of pTFP-hIL2 genes into YGa vector for yeast Saccharomyces cerevisiae.

Since the PCR product contained 40 bp or more of the same sequence as the vector, when introduced into a yeast cell together with the linearized vector, a crossed plasmid vector was produced by crossing within the cell. The transformants formed through intracellular recombination were grown in UD medium (yeast substrate lacking 0.67% amino acid, nutritional supplement lacking 0.77% uracil, 2% glucose, 2% agar) Respectively.

Then, 0.4 ml of acetone was added to 0.6 ml of the supernatant, and the mixture was allowed to stand at -20 ° C for 2 hours, and the protein After centrifugation at 13,000 rpm for 15 minutes, SDS-PAGE gel containing 12% trypsin was applied at 80 V for about 3 hours. After electrophoresis, the cells were stained with decolorization buffer (10% Acetic acid, PTFP2-hIL2, YGa / pTFP3-hIL2, YGa / pTFP4-hIL2, YGa / pTFP5-hIL2, and YGa / pTFP5-hIL2 were added to yeast Y2805 strain, The results of SDS-PAGE analysis of the culture supernatant of each of the two single transformants formed by introducing YGa / pTFP7-hIL2 and YGa / pTFP8-hIL2 are shown in FIG.

The relative expression levels of hIL-2 were compared by SDS-PAGE, and the expression of hIL-2 protein was not detected in pTFP5 , the expression level of pTFP1 was the lowest, and the expression level of hIL-2 protein of pTFP4 was the highest. pTFP1, 4, and 7 were the same genes with different amino acid lengths, but their ability to secrete hIL-2 protein as a protein secretion factor was different. Therefore, even if the same gene is different in size, it acts as another protein secretion fusion factor (FIG. 4).

Example 6 Yeast P. pastoris Of pTFP-interleukin-2

Seven kinds of novel protein secretion fusion factors selected from yeast saccharomyces cerevisiae through Example 4 were constructed from genes derived from Pichia pastoris, Respectively.

To introduce seven kinds of target proteins of pTFP-hIL2 in the YGa vector, which had worked in Saccharomyces cerevisiae, into pKiPastoris, the work was carried out by transferring to a pGAPZ (Invitrogen, USA) vector, which is always running. First, the sense primer CR278 (SEQ ID NO: 26) and the antisense primer CR182 (SEQ ID NO: 27) were used as a template and the YGa / pTFP2-hIL2 plasmid was used as a template, and the sense primer CR279 (SEQ ID NO: 28) and the antisense primer CR182 (SEQ ID NO: 27), the YGa / pTFP3-hIL2 plasmid as a template and the sense primer CR280 (SEQ ID NO: 30), the antisense primer CR182 26) and an antisense primer CR182 (SEQ ID NO: 27), a YGa / pTFP5-hIL2 plasmid as a template, a sense primer CR281 (SEQ ID NO: 30), an antisense primer CR182 The sense primer CR278 (SEQ ID NO: 26) and the antisense primer CR182 (SEQ ID NO: 27), and finally the YGa / pTFP8-hIL2 plasmid as the template, the sense primer CR282 Polymerase chain reaction was carried out using primer CR182 (SEQ ID NO: 27) to introduce EcoRI at the 5 'end and SacII at the 3' end (94 ° C. for 5 minutes, 94 ° C. for 30 seconds, 25 < / RTI > for 1 min at 72 < 0 >C; 1 time for 7 min at 72 < 0 > C).

 The fragments of about 1 kb, 650 bp, 600 bp, 700 bp, 800 bp, 780 bp and 650 bp were recovered in this order, and digested with restriction enzymes EcoR I and Sac II and then ligated with pGAPZ vectors treated with the same restriction enzymes in a TaKaRa, Lt; RTI ID = 0.0 > 16 C < / RTI > for 12 hours using a ligation mix. Each ligated vector was transformed into DH5α and plated on LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% NaCl, 25 μg / ml, 2% agarose gelatin) . After the DNA plasmid was extracted, the plasmid sequences were analyzed to confirm that the target protein was introduced into the pGAPZ vector without any sequence problems.

Seven kinds of constructed pGAPZ vector (pGAPZ / pTFP1-IL2, -PGAPZ / pTFP7-IL2) were linearized with restriction enzyme BlnI and then subjected to electrophoresis in a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) region of Pichia pastoris (1% yeast extract, 2% bactopeptone, 1% glucose, 1% sorbitol) was added to each well and incubated for 2 hours And then cultured at 30 ° C for 3 to 4 days in YPDZ (1% yeast extract, 2% bactopeptone, 2% glucose, 100 ug / ml Zeocin and 2% agar) medium.

When pGAPZ / pTFP5-hIL2 and pGAPZ / pTFP7-hIL2 were transformed, about 200 pKi transformants were obtained in the YPDZ medium, but the sequence was not confirmed on the yeast colony PCR to confirm the target protein. , But not with other pGAPZ / pTFP-hIL-2.

In the case of the transformants into which the remaining pGAPZ / pTFP1-hIL2, pGAPZ / pTFP2-hIL2, pGAPZ / pTFP3-hIL2, pGAPZ / pTFP4-hIL2 or pGAPZ / pTFP8- hIL2 were introduced, three single colonies were each cultured in YPD medium C for 40 hours, and as a control, a transformant expressing hIL-2 as an MFa protein secretion factor derived from Saccharomyces cerevisiae and introduced into Pichia in the same manner as the above pTFP series, A total of seven hIL-2 transformants were constructed using the invitrogen (UST) method recommended by the pPINK-LC vector, a signal peptide kit capable of inducing protein overexpression in the paclitaxel. The expression levels of human interleukin proteins were compared by SDS-PAGE analysis of the experimental group and the control group.

As a result, as shown in Fig. 5, hIL-2 constructed in seven kinds of pPINK-LC vectors of Invitrogen, one of the control groups, was hardly expressed, whereas the expression of Saccharomyces cerevisiae Expression of hIL-2 protein was confirmed in all cases in which hIL-2 was expressed using MFα protein secretion fusion factor and pTFP.

In addition, the function of pTFP identified in Fig. 5 was compared by SDS-PAGE and Western blotting in Saccharomyces cerevisiae and Pichia pastoris. As shown in FIG. 6, when the hIL-2 protein band was confirmed by Western blot, the hIL-2 protein expression of pTFP1-hIL-2, which was the least expressed in Saccharomyces cerevisiae , And the expression of pTFP4-hIL-2 protein was 280% higher than that of the control MFα-hIL-2 in the case of Pichia pastoris. Therefore, even if the same protein secretion fusion factor is used, the secretion inducing ability of the target protein varies depending on the strain used for protein expression.

The present inventors have transfected a transformant prepared by introducing pGAPZ / pTFP1-hIL2, pGAPZ / pTFP2-hIL2, pGAPZ / pTFP3-hIL2 and pGAPZ / pTFP8-hIL2 into Escherichia coli DH5α, respectively, KCTC18302P, KCTC18303P, and KCTC18305P, deposited on June 17, 2014 to the Korean Collection for Type Culture (KCTC) and requested to be transferred to the international deposit under the Budapest Treaty on June 9, 2015 To receive the accession numbers KCTC12833BP, KCTC12834BP, KCTC12834BP, and KCTC12837BP, respectively.

Example 7. P. pastoris Confirmation of the copy number of pTFP-interleukin-2

In order to confirm whether the expression of the three kinds of transformants of pGAPZ / pTFP1-hIL2, pGAPZ / pTFP4-hIL2 and pGAPZ / MFα-hIL2, which were over-expressed in the control group in Example 6, The copy number of the inserted gene on the genomic DNA of the transformant was confirmed.

For this, genomic DNA was recovered by the above-described method, and each genomic DNA was recovered by incubating for 24 hours in YPD. After PCR for 30 minutes at 94 ° C for 30 seconds, A hIL-2 fragment of about 400 bp amplified at 25 ° C for 1 min at 72 ° C was added to a DIG-labeled probe using a DIG labeling kit from Roche, Germany Respectively. In addition, the pGAPZ vector plasmid was digested with restriction enzymes BlnI and EcoRI, and a GAP gene fragment of about 300 bp in size was recovered from the agarose gel, and a probe labeled with DIG was prepared using a Roche kit. Then, three sets of genomic DNAs recovered from pGAPZ / pTFP1-hIL2, pGAPZ / pTFP4-hIL2 and pGAPZ / MFα-hIL2 transformants were treated with restriction enzymes BamHI, EcoRI and SalI in triplicate, and 0.9% agarose The restriction enzyme treatment products were isolated at 100 V for 30 minutes in the gel. The separated agarose gel was subjected to DIG-labeled southern hybridization using Roche DIG, and neutralization solution (0.5M NaOH, 1.5M NaCl) was added twice for 15 min. (1M Tris-HCl, pH 7.5, 1.5M NaCl). After 15 min. treatment twice, the SSC buffer purchased from LPS, Korea was diluted to 10X and the nitrocellulose membrane All DNA was transferred during the day using capillary phenomenon. The DNA transferred to the membrane was fixed with UV, and prehybridization buffer (5X SSC, 2% (w / v), 0.1% (w / v) N-laurylsarcosine, 0.02% ) Was reacted at 42 ° C for 30 minutes so as to be treated uniformly on each membrane. Then, DIG-labeled hIL-2 probe and GAPDH probe, which had been pre-boiled in a hybridization buffer, were added so as to be 100 ng / And reacted with the nitrocellulose membrane at 42 DEG C for 6 hours. Treated twice for 15 min with 2X wash buffer (2X SSC, 0.1% (w / v) SDS) twice in 15 min with 0.5X wash buffer (0.5X SSC, 0.1% (w / v) SDS) (Blocking buffer: 0.1 M maleic acid, 0.15 M NaCl, 1% (w / v) blocking reagent) for 1 hour. Blocking buffer was added to anti-digoxigenin-AP ) Was treated with hIL-2 and GAPDH probes for 30 min to induce immunological detection reaction. Finally, the cells were treated twice with washing buffer (0.1 M maleic acid, 0.15 M NaCl, 0.3% (w / v) Tween 20) for 15 min each time, and the area where the probe and the target protein were bound with NBT / BCIP was visualized , Genomic DNA recovered from pGAPZ / pTFP1-hIL2, pGAPZ / pTFP4-hIL2, and pGAPZ / MFα-hIL2 transformants treated with restriction enzymes BamHI, EcoRI and SalI was amplified by using DIG-labeled hIL-2 and GAP probes The detection results are shown in Fig. From this, it was found that the pGAPZ / pTFP1-hIL2, pGAPZ / pTFP4-hIL2 and pGAPZ / MFα-hIL2 transformants were integrated into the genomic DNA of the nonhomologous region other than the GAPDH gene of each genome In particular, in the case of pGAPZ / pTFP4-hIL2 transformants, the signals detected after the treatment with three kinds of restriction enzymes appeared at the same position, which is the same position as the vector size used at the time of insertion, so that there are two copies side by side It was identified as a phenomenon occurring. From the above results, it was confirmed that the expression amount of hIL-2 of pGAPZ / pTFP4-hIL2 transformant was increased up to 280% compared to the control group because the copy number was one copy more.

Example 8. Comparison of Expression of Human Interleukin-2 by Length of pTFP1

pTFP1 and pTFP4 are the same gene, but their nucleotide sequence lengths are 567 bp and 234 bp, respectively, and thus they function as different protein secretion factors. Therefore, the inventors sought to determine whether the expression level of human interleukin-2 was different when the pTFP1 base sequence was constructed and constructed differently in length according to the characteristics in the base sequence.

First, to find the optimal sequence of pTFP1 capable of maximizing protein expression using pGAPZ / pTFP1-hIL2, the pTFP1 portion was divided into five types, which are shown in FIG. FIG. 9 is a schematic diagram showing a polymerase chain reaction (PCR) and intracellular recombination process for introduction into pGAPZ vector in yeast Pichia pastoris.

(SEQ ID NO: 26) / CR349 (SEQ ID NO: 32) primer using the pGAPZ / pTFP1-hIL2 plasmid shown in the schematic diagram in FIG. 10 as a template and 12 signal peptide portions and 20 linker amino acids from the pTFP1 nucleotide sequence 5 ' 32 pTFP1-1 of the total amino acids made, and a portion where the signal peptide portion and the O-glycosylation site are concentrated from the 5 'end using the CR278 (SEQ ID NO: 26) / CR350 (SEQ ID NO: 33) primer and the linker portion 48 pTFP1-2 of the total amino acids, the signal peptide portion, the O-glycosylation dense portion, one N-glycosylation portion and the linker from the 5 'terminus using the CR278 (SEQ ID NO: 26) / CR351 (SEQ ID NO: 26) / CR352 (SEQ ID NO: 35) primer, and the signal peptide portion, the O-glycosylation dense portion (SEQ ID NO: 26) / CR353 (SEQ ID NO: 36) primer, the sequence of the signal peptide portion, O A total of 138 pTFP1-5 amino acids containing a dense portion of glycosylation, two N-glycosylation moieties, a hydrophobic moiety and a linker were PCR amplified (94 ° C for 5 minutes, 94 ° C for 30 seconds, 55 ° C for 30 seconds , Reaction at 72 ° C for 1 minute 25 times; 72 ° C for 7 minutes once). To amplify the hIL-2 gene to be ligated together with the five PCR fragments, the intact pGAPZ / pTFP1-hIL2 plasmid was used as a template and PCR was performed using primers CR354 (SEQ ID NO: 37) / CR334 (SEQ ID NO: 38) Once for 30 minutes at 94 ° C, for 30 seconds at 55 ° C, for 30 seconds at 72 ° C, once for 7 minutes at 72 ° C).

The primers CR278 (SEQ ID NO: 26) / CR334 (SEQ ID NO: 38) were used as templates and the hIL-2 PCR products amplified with each of pTFP1-1, 1-2, 1-3, PCR was carried out using overlap extension PCR to obtain five PCR products (94 ° C for 5 minutes; 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 25 minutes at 72 ° C for 1 minute).

The pTFP1-1-hIL2, pTFP1-2-hIL2, pTFP1-3-hIL2, pTFP1-4-hIL2 and pTFP1-5-hIL2 PCR products were prepared so that the EcoR I site was present at the 5 'end and the Sac II site was present at the 3' , Digested with restriction enzymes and ligated together with the same restriction enzyme-treated pGAPZ vector (FIG. 9). The constructed vectors were transformed into DH5α and only the plasmid was recovered. The obtained five kinds of vectors (pGAPZ / pTFP1-1-hIL2, pGAPZ / pTFP1-2-hIL2 pGAPZ / pTFP1-3-hIL2 pGAPZ / pTFP1-4-hIL2 , And pGAPZ / pTFP1-5-hIL2) were linearized with Bln I restriction enzyme after sequence identification and inserted into a Pichia pastoris by electroporation (2 mm cuvette, 2000 V voltage, 25 ㎌ capacitance, 200 Ω resistance) (1% yeast extract, 2% bactopeptone, 2% bactopeptone, 1% sorbitol) was added to 1 ml of YPDS medium % Glucose, 100 ug / ml myosin, 2% agar) at 30 캜 for 3 to 4 days. From this, 25 transformants of pTFP1-1, 96 of pTFP1-2, 180 of pTFP1-3, 670 of pTFP1-4, and 159 of pTFP1-5 were obtained, and three transformants per kind were obtained from YPD medium (1% yeast extract, 2% bactopeptone, 2% glucose) for 40 hours, and the degree of hIL-2 expression was observed using SDS-PAGE.

As shown in FIG. 12, the expression levels of hIL-2 in the control pGAPZ / MFα-hIL2, pGAPZ / pTFP1-hIL2 transformants and the above 5 transformants were confirmed. As a result, in the case of containing only the signal peptide as in pTFP1-1, human interleukin-2 could not be secreted. When the sequence from pTFP1-2 to pTFP1-5 was present, the expression of human interleukin- The expression level of pTFP1-hIL-2 was similar to that of original pTFP1-hIL-2. In addition, in all cases except for pTFP1-1, it was confirmed that a larger amount of human interleukin-2 protein was secreted than in the case of using MFα signal peptide. In particular, it was confirmed that overexpression of pTFP4-hIL-2 was overrepresented in the control group.

Example 9. Mass production and purification of pTFP4-interleukin-2

The pGAPZ / pTFP4-hIL2 transformant having overexpression as compared with the control group in Example 6 was used to carry out fed-batch culture in a 5 L fermenter to mass-produce human interleukin-2. Prior to the main culture, the cells were firstly cultured in 50 ml YNB (yeast substrate lacking 0.67% amino acid, 0.5% casamino acid, and 2% glucose) medium and then cultured in 150 ml YEPD liquid medium. And cultured at 30 DEG C for 67 hours.

The cell growth of the Pichia pastoris strain GS115 transformed with the pGAPZ / pTFP4-hIL-2 vector in a 5 L fermenter by fermentation was shown graphically in Fig. 13, and 10 ul of the sample medium was subjected to SDS- PAGE and the results are shown in Fig. As shown in Fig. 14, pGAPZ / pTFP4-hIL2 transformant was normalized. As shown in Fig. 14, high-concentration fermentation showed high intracellular secretion and expression of human interleukin-2 in a complete form similar to that of flask culture.

When a recombinant protein is expressed, protein expression may be increased when a small amount of a non-ionic surfactant that slows the folding of the expressed protein is used. Thus, the inventors of the present invention compared the pTFP4-hIL-2 protein fermentation broth produced by adding 0.2% tween20 and the pTFP4-hIL-2 protein fermentation broth produced without addition of tween20 by fermentation under the same conditions.

Protein fermentation broth of each condition was concentrated using ultrafiltration (molecular weight 10 kDa cut-off) with 20 mM sodium acetate (pH 5.0) and purified with 20 mM sodium acetate (pH 5.0) , The enzyme concentrate was adsorbed on a HiPrap SP FF column, and the enzyme was eluted with a gradient of increasing concentration of NaCl (pH 5.0) from 0 to 1M (FIG. 15).

FIG. 15A is an SDS-PAGE analysis result of an ion exchange chromatography profile and an eluate obtained by purifying pTFP4-hIL-2 protein after fermentation without adding 0.2% tween20. FIG. On the other hand, Fig. 15B shows the result of purifying hIL-2 protein by adding 0.2% of tween20.

16A shows the results of protein SDS-PAGE analysis comparing the fermentation broth with and without tween20 before and after purification. As a result of the purification process, it was confirmed that the recovery rate of hIL-2 protein was higher than that in the case of the addition of 0.2% tween20, as shown in Table 3 below.

Further, to increase the purity of the purified hIL-2 protein, the purified fraction was desalted and subjected to gel filtration (2nd GF) column chromatography twice using superdex 75 prep grade column (16 x 600 mm) -PAGE analysis (Fig. 16B).

A total of 1.71 mg of protein was obtained from the second GF and some of them were used for biological activity analysis using the hIL-2 Quantikine ELISA kit (R & D system).

Example 10. Confirmation of proliferation of EL-4 cell line by hIL-2 purified protein

In order to confirm the biological activity of the hIL-2 purified protein purified in Example 9, the proliferation promoting activity of the EL-4 cell line was measured.

As a result, as shown in FIG. 17, hIL-2 protein in the kit was used as a control and lymphocyte proliferation was confirmed by adding the hIL-2 purified protein to be analyzed in the EL-4 cell line.

That is, it was confirmed that the hIL-2 protein overexpressed by pTFP4 in Pichia pastoris has a biological activity promoting the proliferation of the EL-4 cell line.

From the above results, it was confirmed that the pTFP signal peptide of the present invention induces a much stronger expression of the gene than the conventional MFa signal peptide, and the expression vector containing the gene encoding the secretory protein such as interleukin-2 And it was found that the expression can be induced strongly by the insertion.

From the above description, it will be understood by those of ordinary skill in the art to which the present invention pertains that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Korea Biotechnology Research Institute KCTC18301P 20140617 Korea Biotechnology Research Institute KCTC18302P 20140617 Korea Biotechnology Research Institute KCTC18303P 20140617 Korea Biotechnology Research Institute KCTC18305P 20140617 Korea Biotechnology Research Institute KCTC12833BP 20150609 Korea Biotechnology Research Institute KCTC12834BP 20150609 Korea Biotechnology Research Institute KCTC12835BP 20150609 Korea Biotechnology Research Institute KCTC12837BP 20150609

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel translational fusion partner derived from Pichia pastoris          for secretory production of foreign protein and use thereof <130> KPA140626-KR-P1 <150> KR10-2014-0077554 <151> 2014-06-24 <160> 176 <170> Kopatentin 2.0 <210> 1 <211> 189 <212> PRT <213> Pichia pastoris <220> <221> PEPTIDE &Lt; 222 > (1) .. (189) <223> pTFP1 <400> 1 Met Arg Phe Ile Asn Leu Thr Ile Thr Ser Leu Leu Ala Leu Ala Ser   1 5 10 15 Arg Thr Thr Ala Glu Asp Val Thr Val Thr Gln Leu Val Ser Ser Pro              20 25 30 Thr Ser Ala Ser Glu Ala Gly Asp Trp Ser Ala Asn Trp Val Lys Gly          35 40 45 Phe Pro Ile His Ser Ser Cys Asn Gln Thr Lys Phe Asn Gln Leu Ser      50 55 60 Leu Gly Leu Glu Glu Ala Gln Ile Met Ala Ala His Ala Arg Asp His  65 70 75 80 Thr Leu Arg Tyr Gly Asn Glu Ser Glu Phe Phe Thr Gln Tyr Phe Gly                  85 90 95 Asn Ala Ser Thr Ala Glu Val Ile Gly Trp Phe Ser Val Val Val Asp             100 105 110 Ala Asp Lys Ser Asn Leu Leu Phe Arg Cys Asp Asp Ile Asp Gly Asn         115 120 125 Cys Lys Phe Asp Gly Trp Ala Gly His Trp Arg Gly Glu Asn Gly Ser     130 135 140 Asp Glu Thr Val Val Cys Asp Leu Ser Phe Gln Thr Arg Lys Phe Leu 145 150 155 160 Ala Gln Met Cys Ser Asn Gly Tyr Asn Val Ala Asn Tyr Pro Asn Asn                 165 170 175 Leu Ala Ala Ser Ser Ala Gly Leu Ala Leu Asp Lys Arg             180 185 <210> 2 <211> 567 <212> DNA <213> Pichia pastoris <220> <221> gene <222> (1). (567) <223> pTFP1 <400> 2 atgcgattca ttaacttgac tattacaagt ttacttgcac tggcttccag aacaacagct 60 gaagatgtca ccgtcacaca attggtttct agcccaacct ctgcgtctga agcaggggac 120 tggtctgcta attgggttaa ggggtttcca atacattctt cctgtaacca gactaagttc 180 aaccaactgt ccctgggtct agaagaggct cagatcatgg ccgctcatgc cagagatcac 240 accttgagat atggtaacga gtctgagttc ttcactcaat acttcggtaa tgctagtacc 300 gcagaagtca tcggatggtt ctcagtggtc gttgatgccg acaaatccaa cctgctattc 360 agatgcgatg acatcgacgg aaactgtaaa tttgatggct gggccggtca ctggagaggt 420 gaaaatggat ccgacgaaac tgtcgtttgt gatctttcct tccagaccag gaagttcctt 480 gctcaaatgt gctccaacgg ttataacgtt gccaactacc caaacaacct ggccgcctcc 540 tctgctggcc tcgccttaga taaaaga 567 <210> 3 <211> 69 <212> PRT <213> Pichia pastoris <220> <221> PEPTIDE <222> (1) (69) <223> pTFP2 <400> 3 Met Arg Ile Val Arg Ser Val Ala Ile Ala Ile Ala Cys His Cys Ile   1 5 10 15 Thr Ala Leu Ala Asn Pro Gln Ile Pro Phe Asp Gly Asn Tyr Thr Glu              20 25 30 Ile Ile Val Pro Asp Thr Glu Val Asn Ile Gly Gln Ile Val Asp Ile          35 40 45 Asn His Glu Ile Lys Pro Lys Leu Ala Ala Ser Ala Ser Ala Gly Leu      50 55 60 Ala Leu Asp Lys Arg  65 <210> 4 <211> 207 <212> DNA <213> Pichia pastoris <220> <221> gene &Lt; 222 > (1) <223> pTFP2 <400> 4 atgaggatag taaggagcgt agctatcgca atagcctgtc attgtataac agcgttagca 60 aaccctcaaa tcccttttga cggcaactac accgagatca tcgtgccaga taccgaagtt 120 aacatcggac agattgtaga tattaaccac gaaataaaac ccaaactggc cgcctcggcc 180 tctgctggcc tcgccttaga taaaaga 207 <210> 5 <211> 56 <212> PRT <213> Pichia pastoris <220> <221> PEPTIDE <222> (1). (56) <223> pTFP3 <400> 5 Met Gln Phe Leu Lys Thr Ile Ser Leu Ala Ile Ser Ser Thr Leu Val   1 5 10 15 Ser Ser Ala Val Gly Gly Pro Ile Arg Lys Phe Gly Asn Asp Ser Lys              20 25 30 Gln Cys Thr Asp Ser Ser Phe Tyr His Asn Leu Ala Ala Ser Ala Ser          35 40 45 Ala Gly Leu Ala Leu Asp Lys Arg      50 55 <210> 6 <211> 168 <212> DNA <213> Pichia pastoris <220> <221> gene &Lt; 222 > (1) <223> pTFP3 <400> 6 atgcaattct taaagacaat ttctttggct atcatttcca ctcttgtctc gtctgctgtg 60 ggaggaccta ttagaaaatt tggtaacgac tccaaacagt gcactgattc aagcttctac 120 cacaacctgg ccgcctcggc ctctgctggc ctcgccttag ataaaaga 168 <210> 7 <211> 78 <212> PRT <213> Pichia pastoris <220> <221> PEPTIDE <222> (1) (78) <223> pTFP4 <400> 7 Met Arg Phe Ile Asn Leu Thr Ile Thr Ser Leu Leu Ala Leu Ala Ser   1 5 10 15 Arg Thr Thr Ala Glu Asp Val Thr Val Thr Gln Leu Val Ser Ser Pro              20 25 30 Thr Ser Ala Ser Glu Ala Gly Asp Trp Ser Ala Asn Trp Val Lys Gly          35 40 45 Phe Pro Ile His Ser Ser Cys Asn Gln Thr Lys Phe Asn Gln Leu Pro      50 55 60 Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg  65 70 75 <210> 8 <211> 234 <212> DNA <213> Pichia pastoris <220> <221> gene &Lt; 222 > (1) .. (234) <223> pTFP4 <400> 8 atgcgattca ttaacttgac tattacaagt ttacttgcac tggcttccag aacaacagct 60 gaagatgtca ccgtcacaca attggtttct agcccaacct ctgcgtctga agcaggggac 120 tggtctgcta attgggttaa ggggtttcca atacattctt cctgtaacca gactaagttc 180 aaccaactgc ccctggccgc ctcggcctct gctggcctcg ccttagataa aaga 234 <210> 9 <211> 128 <212> PRT <213> Pichia pastoris <220> <221> PEPTIDE &Lt; 222 > (1) .. (128) <223> pTFP5 <400> 9 Met Ile Phe Asn Leu Lys Thr Leu Ala Ala Val Ala Ile Ser Ile Ser   1 5 10 15 Gln Val Ser Ala Val Ser Ser Leu Gly Phe Ala Leu Gly Asn Lys Asn              20 25 30 Val Asp Gly Thr Cys Lys Tyr Leu Ala Asp Tyr Glu Ala Asp Leu Asp          35 40 45 Thr Ile Arg Gly Gly Ser Glu Ala Val Ala Ile Arg Ala Tyr Ser Ala      50 55 60 Glu Asp Cys Asn Thr Leu Gln Tyr Leu Gly Pro Ala Val Glu Glu Lys  65 70 75 80 Gly Phe Lys Leu Val Leu Ser Val Val Arg Ile Phe Glu Asn Ser Arg                  85 90 95 Ile Gln Lys Ala Ile Thr Ala Met Lys Ile Leu Ser Ala Tyr Gln Gly             100 105 110 Gln Ile Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg         115 120 125 <210> 10 <211> 384 <212> DNA <213> Pichia pastoris <220> <221> gene <222> (1) (384) <223> pTFP5 <400> 10 atgatcttta atcttaaaac actggctgcg gttgcaatct ccatttcaca agtgtctgca 60 gtttcctctc tgggttttgc tctcggaaac aagaacgttg acggaacttg taagtacttg 120 gccgactacg aggccgactt ggatactatt agaggcggct ctgaagccgt tgctattaga 180 gcttattccg ctgaagactg taacacttta caatacctcg gtcctgctgt tgaagagaag 240 ggcttcaaat tagttctatc agtcgtaaga atttttgaaa attcaagaat tcaaaaggcc 300 attacggcca tgaaaatatt aagtgcatat caaggacaaa ttctggccgc ctcggcctct 360 gctggcctcg ccttagataa aaga 384 <210> 11 <211> 108 <212> PRT <213> Pichia pastoris <220> <221> PEPTIDE &Lt; 222 > (1) <223> pTFP7 <400> 11 Met Arg Phe Ile Asn Leu Thr Ile Thr Ser Leu Leu Ala Leu Ala Ser   1 5 10 15 Arg Thr Thr Ala Glu Asp Val Thr Val Thr Gln Leu Val Ser Ser Pro              20 25 30 Thr Ser Ala Ser Glu Ala Gly Asp Trp Ser Ala Asn Trp Val Lys Gly          35 40 45 Phe Pro Ile His Ser Ser Cys Asn Gln Thr Lys Phe Asn Gln Leu Ser      50 55 60 Leu Gly Leu Glu Glu Ala Gln Ile Met Ala Ala His Ala Arg Asp His  65 70 75 80 Thr Leu Arg Tyr Gly Asn Glu Ser Glu Phe Phe Thr Gln Tyr Leu Ala                  85 90 95 Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg             100 105 <210> 12 <211> 324 <212> DNA <213> Pichia pastoris <220> <221> gene &Lt; 222 > (1) <223> pTFP7 <400> 12 atgcgattca ttaacttgac tattacaagt ttacttgcac tggcttccag aacaacagct 60 gaagatgtca ccgtcacaca attggtttct agcccaacct ctgcgtctga agcaggggac 120 tggtctgcta attgggttaa ggggtttcca atacattctt cctgtaacca gactaagttc 180 aaccaactgt ccctgggtct agaagaggct cagatcatgg ccgctcatgc cagagatcac 240 accttgagat atggtaacga gtctgagttc ttcactcaat acctggccgc ctcggcctct 300 gctggcctcg ccttagataa aaga 324 <210> 13 <211> 67 <212> PRT <213> Pichia pastoris <220> <221> PEPTIDE <222> (1) (67) <223> pTFP8 <400> 13 Met Asn Pro Ser Ser Leu Ile Leu Leu Ala Leu Ser Ile Gly Tyr Ser   1 5 10 15 Ile Ala Glu Ser Asn Phe Ser Phe Lys Pro Ser Lys Leu Pro Leu Lys              20 25 30 Lys His Arg Asp Ser Ser Ser Pro His Glu Arg Phe Leu Lys Arg Asp          35 40 45 Gly Pro Tyr Gln Pro Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu      50 55 60 Asp Lys Arg  65 <210> 14 <211> 201 <212> DNA <213> Pichia pastoris <220> <221> gene &Lt; 222 > (1) .. (160) <223> pTFP8 <400> 14 atgaacccta gcagcttaat tctacttgca ctcagcattg gctactccat tgctgagtca 60 aatttctctt tcaaacccag caagttacct ctcaaaaaac atcgtgattc ttcttccccg 120 catgaacgat ttcttaaacg agatggaccc tatcagccgc tggccgcctc ggcctctgct 180 ggcctcgcct tagataaaag a 201 <210> 15 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> GalSfi1 primer <400> 15 gtaagaattt ttgaaaattc aagaattcaa aaggccatta cggc 44 <210> 16 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> SfiB3'N6 primer <400> 16 attgaccttt tatctagggc cgaggcggcc agnnnnnn 38 <210> 17 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> SfiB3 'primer <400> 17 ctagtttcgt ttgtcattga ccttttatct agggccgagg cggcc 45 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Gal100 primer <400> 18 gtatatggtg gtaatgccat g 21 <210> 19 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> LDKR42 primer <400> 19 tcttttatct aaggcgaggc cagcagaggc cgaggcggcc 40 <210> 20 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> LNK39 primer <400> 20 ggccgcctcg gcctctgctg gcctcgcctt agataaaaga 40 <210> 21 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> CR139 primer <400> 21 gtttcgtttg tcattgaagt tagtgttgag atgatgc 37 <210> 22 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> CR138 primer <400> 22 gcatcatctc aacactaact tcaatgacaa acgaaactag 40 <210> 23 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> CR142 primer <400> 23 ccccatacgg tgtcatttgg gttgtattga aagtac 36 <210> 24 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> CR143 primer <400> 24 cactccgttc aagtcgactc aagtcagtgt tgagatg 37 <210> 25 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> GT50R primer <400> 25 gtcattatta aatatatata tatatatatt gtcactccgt tcaagtcgac 50 <210> 26 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> CR278 primer <400> 26 ggaattccat gcgattcatt aacttgacta ttac 34 <210> 27 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CR182 primer <400> 27 tccccgcggg gatcaagtca gtgttgagat g 31 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CR279 primer <400> 28 ggaattccat gaggatagta aggagcgtag 30 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> CR280 primer <400> 29 ggaattccat gcaattctta aagacaattt ctttgg 36 <210> 30 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> CR281 primer <400> 30 ggaattccat gatctttaat cttaaaacac tggc 34 <210> 31 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CR282 primer <400> 31 ggaattccat gaaccctagc agcttaattc 30 <210> 32 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> CR349 primer <400> 32 ctaaggcgag gccagcagag gaggcggcag ctgttgttct ggaagcc 47 <210> 33 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> CR350 primer <400> 33 ctaaggcgag gccagcagag gaggcggcag acgcagaggt tgggctag 48 <210> 34 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> CR351 primer <400> 34 ctaaggcgag gccagcagag gaggcggcca gggacagttg gttgaac 47 <210> 35 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> CR352 primer <400> 35 ctaaggcgag gccagcagag gaggcggcct cagactcgtt accatatc 48 <210> 36 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> CR353 primer <400> 36 ctaaggcgag gccagcagag gaggcggcgt cgatgtcatc gcatctg 47 <210> 37 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> CR354 primer <400> 37 ctctgctggc ctcgccttag ataaagagc acctacttca agttctaca 49 <210> 38 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CR334 primer <400> 38 ggcaaatggc attctgacat cc 22 <210> 39 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 1F primer <400> 39 caaaaggcca ttacggccat gaggatagta aggagc 36 <210> 40 <211> 17 <212> DNA <213> Artificial Sequence <220> 1R primer <400> 40 tagataatca tcagagc 17 <210> 41 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> 2F primer <400> 41 caaaaggcca ttacggccat gaagaattta gctaaac 37 <210> 42 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 2R primer <400> 42 gaatttgctc aggttggc 18 <210> 43 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> 3F primer <400> 43 caaaaggcca ttacggccat gaaacttcag ttatttac 38 <210> 44 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 3R primer <400> 44 tgcaagagta gctccata 18 <210> 45 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 4F primer <400> 45 caaaaggcca ttacggccat gtcattctct tccaac 36 <210> 46 <211> 17 <212> DNA <213> Artificial Sequence <220> <222> 4R primer <400> 46 gacaccaaca tctggac 17 <210> 47 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 5F primer <400> 47 caaaaggcca ttacggccat gtggatcttg tggct 35 <210> 48 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 5R primer <400> 48 ctgatcgttg ttacaattc 19 <210> 49 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 6F primer <400> 49 caaaaggcca ttacggccat gaaaatatta agtgc 35 <210> 50 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 6R primer <400> 50 gaatttgtcc ttgatatg 18 <210> 51 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> 7F primer <400> 51 caaaaggcca ttacggccat gaaattgttg aactttc 37 <210> 52 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 7R primer <400> 52 caactcatct ttcacgac 18 <210> 53 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 8F primer <400> 53 caaaaggcca ttacggccat gagaccagtg ctttcg 36 <210> 54 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 8R primer <400> 54 atctgggacg tcataac 17 <210> 55 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 9F primer <400> 55 caaaaggcca ttacggccat gtttgtgatc cagctg 36 <210> 56 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 9R primer <400> 56 gctactaaaa taactggc 18 <210> 57 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 10F primer <400> 57 caaaaggcca ttacggccat gtttaaatct ctgtgc 36 <210> 58 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 10R primer <400> 58 gtagttgtta gtctcttc 18 <210> 59 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 11F primer <400> 59 caaaaggcca ttacggccat gttgtccatt ttaag 35 <210> 60 <211> 18 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 11R primer <400> 60 caaaccgtaa ttgttttc 18 <210> 61 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 12F primer <400> 61 caaaaggcca ttacggccat gcaagttaaa tctatc 36 <210> 62 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 12R primer <400> 62 aacggcagca ccgttgg 17 <210> 63 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 13F primer <400> 63 caaaaggcca ttacggccat gatggtgttt ggatgc 36 <210> 64 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 13R primer <400> 64 aacgtggcca gtcagcc 17 <210> 65 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 14F primer <400> 65 caaaaggcca ttacggccat gctcttcgga ctaatt 36 <210> 66 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 14R primer <400> 66 gcgtattgct aacagtc 17 <210> 67 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> 15F primer <400> 67 caaaaggcca ttacggccat gagattactt cacatttc 38 <210> 68 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 15R primer <400> 68 atctaccagt ggtaacac 18 <210> 69 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 16F primer <400> 69 caaaaggcca ttacggccat gtggagacct cttgtt 36 <210> 70 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 16R primer <400> 70 tccagatcct tgacatg 17 <210> 71 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> 17F primer <400> 71 caaaaggcca ttacggccat gatctttaat cttaaaac 38 <210> 72 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 17R primer <400> 72 taactgtctg gaactgtc 18 <210> 73 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 18F primer <400> 73 caaaaggcca ttacggccat gtttgagaag agtaa 35 <210> 74 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 18R primer <400> 74 ggtgaaagta ccggtccaa 19 <210> 75 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> 19F primer <400> 75 caaaaggcca ttacggccat gaaatatttg ccactcg 37 <210> 76 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 19R primer <400> 76 agagatggta ccagaac 17 <210> 77 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 20F primer <400> 77 caaaaggcca ttacggccat gctatcaact atctt 35 <210> 78 <211> 19 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 20R primer <400> 78 cacagtaatg agctcaaag 19 <210> 79 <211> 35 <212> DNA <213> Artificial Sequence <220> <21> 21F primer <400> 79 caaaaggcca ttacggccat gctgtcgtta aaacc 35 <210> 80 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 21R primer <400> 80 gacctctctc ttcagctt 18 <210> 81 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 22F primer <400> 81 caaaaggcca ttacggccat gatccgattg ttggc 35 <210> 82 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 22R primer <400> 82 gtggcaacca ttggagaa 18 <210> 83 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> 23F primer <400> 83 caaaaggcca ttacggccat gaacttgtac ctaattac 38 <210> 84 <211> 17 <212> DNA <213> Artificial Sequence <220> <233> 23R primer <400> 84 ccagtggtac tctttat 17 <210> 85 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 24F primer <400> 85 caaaaggcca ttacggccat gcagtttgga aaggt 35 <210> 86 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 24R primer <400> 86 ccattcactg aagtcag 17 <210> 87 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 25F primer <400> 87 caaaaggcca ttacggccat gaagctctcc accaa 35 <210> 88 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 25R primer <400> 88 aacaatattt ccacgagg 18 <210> 89 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 26F primer <400> 89 caaaaggcca ttacggccat gaaaattgcc cagttg 36 <210> 90 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 26R primer <400> 90 gtaagtcatc acgttcc 17 <210> 91 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 27F primer <400> 91 caaaaggcca ttacggccat gaaatcaagt tggaa 35 <210> 92 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 27R primer <400> 92 tctcggtttg ttgaaaa 17 <210> 93 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 28F primer <400> 93 caaaaggcca ttacggccat gttgagactt ctgac 35 <210> 94 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 28R primer <400> 94 aatcgattcg tatttag 17 <210> 95 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 29F primer <400> 95 caaaaggcca ttacggccat gctatatttg gtgac 35 <210> 96 <211> 20 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 29R primer <400> 96 cggttcttgg gcaaactcat 20 <210> 97 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 30F primer <400> 97 caaaaggcca ttacggccat gaaccctagc agctta 36 <210> 98 <211> 17 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 30R primer <400> 98 acctgaagtc acgtatg 17 <210> 99 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 31F primer <400> 99 caaaaggcca ttacggccat gttgcccatc cgctta 36 <210> 100 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 31R primer <400> 100 ttctccctca ctatcgca 18 <210> 101 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 32F primer <400> 101 caaaaggcca ttacggccat gctgggtttc aaggac 36 <210> 102 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 32R primer <400> 102 aacaggatag ccagccat 18 <210> 103 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 33F primer <400> 103 caaaaggcca ttacggccat gtttggaaag aggag 35 <210> 104 <211> 18 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 33R primer <400> 104 atgtccagat ttaagcag 18 <210> 105 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 34F primer <400> 105 caaaaggcca ttacggccat gtaccaggcg ttgttg 36 <210> 106 <211> 17 <212> DNA <213> Artificial Sequence <220> <34> 34R primer <400> 106 tgcattaagc tgcactg 17 <210> 107 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 35F primer <400> 107 caaaaggcca ttacggccat gctctataag accacc 36 <210> 108 <211> 17 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > <400> 108 gtgaacggca tagcatg 17 <210> 109 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 36F primer <400> 109 caaaaggcca ttacggccat gcgattcatt aacttg 36 <210> 110 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 36R primer <400> 110 agcacagtgg acttcgccat 20 <210> 111 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 37F primer <400> 111 caaaaggcca ttacggccat gaaatcggtt atttgg 36 <210> 112 <211> 19 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 37R primer <400> 112 tggctgatag tacttatac 19 <210> 113 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 38F primer <400> 113 caaaaggcca ttacggccat gtttaatagc ttggc 35 <210> 114 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 38R primer <400> 114 cgtgtagaaa tcgttaa 17 <210> 115 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 39F primer <400> 115 caaaaggcca ttacggccat gttagttgct gttgc 35 <210> 116 <211> 17 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 39R primer <400> 116 gactaaaaac agatcct 17 <210> 117 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 40F primer <400> 117 caaaaggcca ttacggccat gttttggtta ttggt 35 <210> 118 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 40R primer <400> 118 gtaggcccat cggcgtt 17 <210> 119 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> 41F primer <400> 119 caaaaggcca ttacggccat gaaatttttt tactttgc 38 <210> 120 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 41R primer <400> 120 gtagtttcct ccgtcaac 18 <210> 121 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 42F primer <400> 121 caaaaggcca ttacggccat gaaatttcct gtgcc 35 <210> 122 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 42R primer <400> 122 gacagtgtaa gtggtctc 18 <210> 123 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 43F primer <400> 123 caaaaggcca ttacggccat gaagctcgct gcact 35 <210> 124 <211> 17 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 43R primer <400> 124 attgtaaaac gaaccag 17 <210> 125 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 44F primer <400> 125 caaaaggcca ttacggccat gctgaataga gtgttg 36 <210> 126 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 44R primer <400> 126 cgaggtcgaa ttagagct 18 <210> 127 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 45F primer <400> 127 caaaaggcca ttacggccat gaatttaact ttgatc 36 <210> 128 <211> 18 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 45R primer <400> 128 tttgttcaat ttggtgta 18 <210> 129 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 46F primer <400> 129 caaaaggcca ttacggccat ggtttcttta acaag 35 <210> 130 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 46R primer <400> 130 aagaagagca aactcctc 18 <210> 131 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 47F primer <400> 131 caaaaggcca ttacggccat gaagctatta gacgg 35 <210> 132 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 47R primer <400> 132 agccagttca atacctc 17 <210> 133 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 48F primer <400> 133 caaaaggcca ttacggccat gttgaaggat cagttc 36 <210> 134 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> 48R primer <400> 134 atcggtgacc atcttg 16 <210> 135 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> 49F primer <400> 135 caaaaggcca ttacggccat gatgattagt gcgtttg 37 <210> 136 <211> 17 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 49R primer <400> 136 accttcgaaa ctggatc 17 <210> 137 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 50F primer <400> 137 caaaaggcca ttacggccat gcaattcaac tggaat 36 <210> 138 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 50R primer <400> 138 atctttgttc gtcaattc 18 <210> 139 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 51F primer <400> 139 caaaaggcca ttacggccat gggattgttg gagaag 36 <210> 140 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 51R primer <400> 140 cttatccgga aacttaga 18 <210> 141 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 52F primer <400> 141 caaaaggcca ttacggccat gatattacac acctat 36 <210> 142 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> 52R primer <400> 142 accaccttca ccacaa 16 <210> 143 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 53F primer <400> 143 caaaaggcca ttacggccat gcaattctta aagac 35 <210> 144 <211> 18 <212> DNA <213> Artificial Sequence <220> <53> 53R primer <400> 144 ttcaaaggac caagtaac 18 <210> 145 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 54F primer <400> 145 caaaaggcca ttacggccat gaagatctct accat 35 <210> 146 <211> 19 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 54R primer <400> 146 cttggtggcc tctggatca 19 <210> 147 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 55F primer <400> 147 caaaaggcca ttacggccat gttcaaagga gctagg 36 <210> 148 <211> 18 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 55R primer <400> 148 tgagttgtga tgtaatgc 18 <210> 149 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 56F primer <400> 149 caaaaggcca ttacggccat gaaagggatc aagttc 36 <210> 150 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 56R primer <400> 150 ttctctaaaa gcttgcg 17 <210> 151 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 57F primer <400> 151 caaaaggcca ttacggccat gagcaccctg acattg 36 <210> 152 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 57R primer <400> 152 tattaccgtc gtttctg 17 <210> 153 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 58F primer <400> 153 caaaaggcca ttacggccat ggttttgata caaaa 35 <210> 154 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 58R primer <400> 154 atacaggaag tagatgg 17 <210> 155 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> 59F primer <400> 155 caaaaggcca ttacggccat gttatcccta acaaaac 37 <210> 156 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 59R primer <400> 156 agtagaggtc agtccaatg 19 <210> 157 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 60F primer <400> 157 caaaaggcca ttacggccat gaaactactc ttacc 35 <210> 158 <211> 18 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 60R primer <400> 158 caaccctcca gagaaaac 18 <210> 159 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 61F primer <400> 159 caaaaggcca ttacggccat gattaattta aactcc 36 <210> 160 <211> 18 <212> DNA <213> Artificial Sequence <220> <201> 61R primer <400> 160 tggagggaca gtaactgt 18 <210> 161 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 62F primer <400> 161 caaaaggcca ttacggccat ggtttcactg cgatc 35 <210> 162 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 62R primer <400> 162 aggctcgtca gatgtagt 18 <210> 163 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 63F primer <400> 163 caaaaggcca ttacggccat gaagttacta tccttg 36 <210> 164 <211> 19 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 63R primer <400> 164 taattcgtcg tgatttttg 19 <210> 165 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 64F primer <400> 165 caaaaggcca ttacggccat gagacaagtt gattg 35 <210> 166 <211> 18 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > <400> 166 cttgtacttt ttatcaag 18 <210> 167 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 65F primer <400> 167 caaaaggcca ttacggccat gaagtcgtta ctgct 35 <210> 168 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> The 65R primer <400> 168 caattccctt agttggg 17 <210> 169 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 66F primer <400> 169 caaaaggcca ttacggccat gttggtcgcc tggttt 36 <210> 170 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 66R primer <400> 170 aggggatata aacttattc 19 <210> 171 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 67F primer <400> 171 caaaaggcca ttacggccat gaagatatcc gctct 35 <210> 172 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 67R primer <400> 172 agttggtata accatgt 17 <210> 173 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 68F primer <400> 173 caaaaggcca ttacggccat gaaatctcaa cttatc 36 <210> 174 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 68R primer <400> 174 gttgggagcc ttcttgtt 18 <210> 175 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 69F primer <400> 175 caaaaggcca ttacggccat gcaattcaac agtgtc 36 <210> 176 <211> 18 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 69R primer <400> 176 ggctccttta gaaccagt 18

Claims (11)

SEQ ID NO: 1 amino acid sequence or a 1 to 48 times consisting of a fragment of the amino acid sequence of SEQ ID NO: 1 comprising the amino acid of, Pichia secreting produce the desired protein in my process (Saccharomyces) in microorganisms (Pichia) in or Saccharomyces As protein fusion factors,
Interferon, interferon, interferon, colony stimulating factor (GM-CSF), interleukin-2 (interleukin-2), blood factor VII, blood factor VIII, blood factor IX, immunoglobulin, cytokine, ), TGF-a, TGF-beta, follicle-stimulating hormone (TGF-beta), insulin, tumor necrosis factor (TNF), EGF, platelet derived growth factor (PDGF), phospholipase- Calcitonin Gene Related Peptide (CGPR), enkephalin, somatomedin, erythropoietin, erythropoietin, thyroid-stimulating hormone, antidiuretic hormone, pigment hormone, parathyroid hormone, luteinizing hormone, calcitonin, (GHPR), thymic humoral factor (THF), asparaginase, arginine, and the like. Gina An antiobesity agent such as arginine deaminase, adenosine deaminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, ukase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, Wherein the protein fusion factor is selected from the group consisting of cytidase, glucocerebrosidase, and glucuronidase.
The protein fusion factor according to claim 1, wherein the microorganism is Saccharomyces cerevisiae or Pichia pastoris .
2. The protein fusion factor according to claim 1, wherein the target protein is interleukin-2.
A polynucleotide encoding the protein fusion factor of claim 1.
5. The polynucleotide of claim 4, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2.
5. An expression vector comprising the polynucleotide of claim 4.
7. An expression vector according to claim 6, wherein said vector comprises a nucleic acid encoding a target protein,
Interferon, interferon, interferon, colony stimulating factor (GM-CSF), interleukin-2 (interleukin-2), blood factor VII, blood factor VIII, blood factor IX, immunoglobulin, cytokine, ), TGF-a, TGF-beta, follicle-stimulating hormone (TGF-beta), insulin, tumor necrosis factor (TNF), EGF, platelet derived growth factor (PDGF), phospholipase- Calcitonin Gene Related Peptide (CGPR), enkephalin, somatomedin, erythropoietin, erythropoietin, thyroid-stimulating hormone, antidiuretic hormone, pigment hormone, parathyroid hormone, luteinizing hormone, calcitonin, (GHPR), thymic humoral factor (THF), asparaginase, arginine, and the like. Gina An antiobesity agent such as arginine deaminase, adenosine deaminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, ukase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, Wherein the expression vector is selected from the group consisting of cytidase, glucocerebrosidase, and glucuronidase.
A transformant comprising the expression vector of claim 6.
9. The transformant according to claim 8, wherein the transformant is the accession number KCTC18301P.
Article comprising the step of culturing a My process (Saccharomyces) in a microorganism of the genus Saccharomyces or the vector of claim 7 is introduced into Pichia (Pichia) in the culture medium, the desired protein production method.
11. The method of claim 10, wherein the microorganism is Saccharomyces cerevisiae or Pichia pastoris.

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