KR20050076139A - Translational fusion partners for the secretory production of proteins - Google Patents

Abstract

The present invention provides a high-speed translational fusion partner (TFP) capable of expressing and secreting non-producible proteins that are difficult to produce recombinantly from various gene sources. Protein secretion induction obtained using the screening technique relates to a fusion factor.

Description

Translational fusion partners for the secretory production of proteins

The present invention relates to a protein fusion factor obtained by using a technique for ultrafast selection of a translational fusion partner (TFP) from various gene sources capable of expressing and secreting a protein that is difficult to produce recombinantly in yeast. .

In order to analyze the genome sequence information obtained from the recent human genome project and the functions of various proteins revealed in the genome unit, and to produce pharmacologically important protein products for humans, development of a highly efficient protein production system using recombinant microorganisms is required. When selecting an expression system to produce recombinant protein derived from higher organisms such as human body, growth characteristics of the host cell, protein expression level, intracellular and external expression potential, post-translational modification possibility, and biological expression of the expressed protein Various factors should be considered, such as activity and the use of expression proteins. As a representative microbial gene expression system, E. coli and yeast systems are mainly used.E. coli has a lot of expression systems and a high expression rate of foreign proteins. It is impossible to be completely secreted by the cell culture medium, and it is impossible to fold the protein with many disulfide bonds, and it is pointed out that it is produced in the form of insoluble protein such as inclusion body. (Makrides, Microbial Rev., 1996, 60, 512). In addition, since most of the high medicinal proteins associated with diseases in human proteins are glycoproteins or membrane proteins, they must require glycosylation or complete three-dimensional structure through disulfide bonds to have full activity. In E. coli, production is impossible and eukaryotic expression systems such as yeast are essential.

Eukaryotic microbial yeast Saccharomyces cerevisiae (Generally Recognized As Safe) GRAS (Generally Recognized As Safe) has proven to be safe for humans, easy to genetically engineer, various expression systems have been developed and easy to mass cultivate. In addition, when recombinant production of higher cell-derived proteins such as human proteins, it provides a secretion function to secrete proteins out of the cell and post-translational modification of proteins such as sugar chains. Extracellular secretion is possible by artificially fusion of the secretion signal of the protein with the target protein, and the process of folding the protein, forming disulfide bonds and adding sugar chains through the secretion process of the protein leads to a biologically complete recombinant protein. It offers the advantage of producing It is also very economical because proteins with biological activity can be obtained directly from the medium and do not require economically inefficient cell grinding or refolding steps (Eckart and Bussineau, Curr. Opin. Biotechnol., 1996, 7 , 525).

However, despite the many advantages described above, it is pointed out as a problem of the current technology regarding the human protein secretion system using the yeast Saccharomyces cerevisiae, which is not produced at all or several grams / liter depending on the type of human protein. It is difficult to predict secretion productivity because the back secretion rate is more than a thousand times different. When exogenous protein can produce up to several grams / liter of secretion, it seems that there is sufficient economical efficiency in terms of secretion productivity, but it is low in secretion efficiency depending on the type of protein, and especially to produce high value-added human pharmaceutical protein. It is often difficult to express and secrete. In order to solve this problem, a lot of researches on the secretion factors involved in the secretion of proteins. Overexpression of BiP ( KAR2 ), a secretion factor to help fold newly synthesized proteins in endoplasmic reticulum (Robinson et al., Biotechnol. Prog ., 1996, 271, 10017) and overexpression of PDI (protein disulfide isomerase) to help form cysteine bonds Methods for chaperone (Robinson et al., Bio / Technology, 1994, 12, 381; Schultz et al., Ann. NY Acad. Sci., 1994, 721, 148; Hayano et al., FEBS Lett., 1995, 377, 505) Research and development of fusion factors that induce secretion and a lot of research to enhance the secretion through the fusion with proteins that are well secreted originally (Gouka et al., Appl. Microbiol. Biotechnol., 1997, 47, 1). This method has been recognized as a very successful method to enhance the secretion of foreign proteins to date. Although the research on the molecular mechanism of the fusion technology is still insufficient, experimentally, this fusion technology facilitates the transport of proteins and helps the protein folding. It is known to improve the limitations in the translation and post-translational stages of the.

Kjeldsen et al. (Protein Expr. Purif., 1997, 9, 331) fused synthetic insulins prepared by theoretical basis to insulin to efficiently secrete insulin or insulin precursor (IP) from yeast. By improving the secretion rate of insulin, the synthesis leader was added with a glycosylation site (N-glycosylation site) and BiP recognition site to induce the protein to be properly folded by extending the retention time in the endoplasmic reticulum. The addition of additional sugar chain sites to the synthetic leader also reported a significant increase in insulin secretion in Aspergillus niger and Saccharomyces cerevisiae (Kjeldsen et al., Protein Expr. Purif., 1998, 14, 309). . Similar results were reported for the expression of Aspergillus awamori (Ward et al., Bio / Technology, 1989, 8, 435) and hydrophobic cutinase in yeast (Sagt et al., Appl. Environ. Microbiol. 2000, 66 , 4940). This was due to the introduction of glycosylation sites to increase the solubility in the endoplasmic reticulum of the recombinant protein and to induce folding of the protein, resulting in increased secretion.

Originally, proteins that secrete well as fusion partners include fusion expression with Aspergillus niger's glucoamylase and bovine prochymosin (Ward et al., Bio / Technology, 1989, 8, 435), Porcine pancreatic phospholipase A2 (Roberts et al., Gene, 1992, 122, 155), human interleukin-6 (Contreras et al., Bio / Technology 1991, 9, 378; Broekhuijsen et al., J. Biotechnol., 1993, 31, 135), hen egg-white lysozyme from chickens (Jeenes et al., FEMS Microbiol Lett, 1993, 107, 267) and human lactoferrin (Ward et al. , Bio / Technology, 1995, 13, 498). Secretion rate increased from 5 to 1000 times, depending on the protein. In yeast, secretion of human growth hormone and granulocyte colony-stimulatinf factor (G-CSF) is achieved by using 24 amino-terminal amino acids of human interleukin-1 beta as fusion partners. Increased (Lee et al., Biotechnol. Prog., 1999, 15, 884). Human interleukin-1beta is known to be secreted without a special secretion signal (Muesch et al., Trends Biochem. Sci., 1990, 15, 86) and has been reported to be highly effective recombinant secretion production in yeast (Baldari et al., Protein Eng. , 1987, 1, 433). It has also been reported that a fusion partner originally possessed by the protein is necessary for proper folding of the protein (Takahashi et al., Appl Microbiol. Biotechnol., 2001, 55, 454). For expression in Seth cerevisiae, the pre-pro-leader sequence of mating factor alpha derived from Saccharomyces was expressed by binding to a gene corresponding to the mature protein of ROL. ROL was not secreted at all, but when the pro sequence of ROL itself was combined, it was properly secreted. This showed that the pro sequence of the ROL itself is absolutely necessary for the folding of its protein.

As shown in the above results, various secretion factors have been developed to induce secretion of recombinant proteins. However, the developed secretion factors are effective in promoting the secretion of specific proteins, but there was a problem that cannot be applied uniformly to all proteins. Dorner et al. Reported that overexpression of BiP in CHO cells resulted in decreased protein secretion (Dorner et al., EMBO J., 1992, 11, 1563), whereas increased protein secretion increased when BiP expression was reduced (Dorner). Et al., Mol. Cell Biol., 1988, 8, 4063). In yeast, overexpression of KAR2 (BiP) did not improve secretion in the case of plant thaumatin (Harmsen et al., Appl. Microbiol. Biotechnol., 1996, 46, 365). Overexpression of BiP in baculovirus increased the amount of soluble antibodies in cell extracts, but did not increase the secretion efficiency of antibodies (Hsu et al., Protein Expr. Purif., 1994, 5, 595). Overexpression of another secretion factor, Fall Days ( PDI ), overexpressed Fall Days in Aspergillus niger but did not increase glucoamylase secretion (Wang and Ward, Curr. Genet. 2000, 37, 57). . In addition, in the case of secretion induction using a protein fusion partner has been reported a problem that the secretion efficiency is enhanced only to a specific protein.

As can be seen from the above results, there have been many studies on the effects of secretion factors, but the secretion factors developed due to the different effects on the secretion according to the type of protein has a problem that can not be applied to all proteins. Therefore, in order to maximize each protein secretion desired, a technique for selecting an optimal secretion factor that can be specifically applied to the protein of interest is required. The present inventors have completed the present invention by developing a technique for selecting an optimal secretion fusion factor at high speed in the genome unit according to the type of recombinant protein.

Accordingly, an object of the present invention is to find a suitable protein fusion factor (TFP) from yeast genome and various gene sources that can strongly induce protein production in proteins with low expression rate in yeast that cannot be economically mass-produced. It is to provide a method and a method that can promote the secretion production of a protein that is difficult to express using the method.

In one embodiment, the present invention binds a poorly expressed target protein gene (X) to an autoselection reporter gene (R) to produce a fusion of XR, and an XR fusion protein through another gene fusion to the fusion of XR. The present invention relates to a method for selecting a protein fusion factor (TFP) from the genetic library that induces secretion out of cells.

Preferably, the present invention

(1) preparing an automatic selection vector comprising a fusion gene (X-R) of an auto-selection reporter gene (R) linked in-frame with a gene (X) of a poorly expressed target protein;

(2) preparing a protein fusion factor library by linking genomic DNA or cDNA including a translational fusion partner (TFP) to induce secretion of the non-expressing fusion protein (X-R) with an automatic selection vector;

(3) detecting the activity of the reporter protein by transforming the protein fusion factor library into cells in which the reporter gene is not active; And

(4) separating the gene from the transformed cell showing the activity of the reporter protein, and analyzing the characteristics of the protein fusion factor, and relates to a method for selecting a custom protein fusion factor for the production of a poorly expressed protein.

More preferably, the present invention

(1) preparing a yeast mutant strain which deletes INV2 (I), which is an invertase gene owned by the yeast, for use as a reporter gene for developing an automatic selection system using yeast invertase;

(2) Yeast HTS, an automatic selection vector containing genes (XI) in which expression is regulated by the yeast GAL10 promoter and invertase (I) gene and non-expressive gene (X) are fused in-frame (high throughput selection) vector (pYHTS-F0, pYHTS-F1 and pYHTS-F2) preparation step;

(3) preparing a protein fusion factor library in the pYHTS vector from the yeast genome capable of secreting a fusion gene (X-I) of invertase and non-expressing protein;

(4) transforming the prepared library to the yeast prepared in step (1) and automatically selecting in a medium containing sucrose as a single carbon source;

(5) culturing the yeast grown in sucrose medium to identify the protein secreted into the medium; And

(6) The present invention relates to a method for selecting TFP for the production of refractory proteins, the method comprising separating genes from yeast and analyzing protein fusion factor properties.

When expressing in yeast by fusing the difficult-recombinant and difficult to express recombinant protein (X) and invertase (I), secretion of the normally inverted (I) secreted by the fused non-expressing protein (X) is thus suppressed. In medium containing sucrose as a single carbon source, yeast does not grow or the growth is very delayed depending on the degree of hard expression. However, the cells grow rapidly in sucrose medium when introducing efficient protein fusion factors that can induce the expression and secretion of X-I. Using this feature, a protein fusion factor library obtained from various genomes is additionally fused to a protein (XI) in which a poorly expressed protein (X) and an invertase (I) are fused to prepare in the form of TFP-XI or XI-TFP. Then, by introducing into yeast, expressing and rapidly growing cells in sucrose medium, TFP can be selected at a very high speed from various genome libraries that are most suitable for the non-expressing protein.

Specifically, the present inventors have confirmed that the invertase can be used as a marker of an automatic selection system by preparing a mutant strain lacking the yeast invertase and expressing a protein fused with invertase in a yeast strain lacking the invertase gene. It was. Subsequently, pYHTS-F0, F1, and F2, protein fusion factor autoselection vectors using human interleukin-2, a non-expressing protein, were prepared, and a protein fusion factor library was prepared by linking the cleaved chromosomal DNA of yeast origin. Refractory Proteins Protein fusion factor proteins TFP-1, TFP-2, TFP-3 and TFP-4 suitable for human interleukin-2 were identified.

In the present invention, a "translational fusion partner (TFP)" refers to a gene that is fused with a gene encoding a poorly expressed protein to induce secretory production of the poorly expressed protein. In addition, "protein fusion factor protein" means a protein of the amino acid sequence encoded by the protein fusion factor gene as described above. Specifically, TFP-1, TFP-2, TFP-3, TFP-4 and the like can be enumerated.

In the present invention, "automatic screening system using invertase" means that the yeast requires an enzyme called invertase, which is a protein encoded by the yeast INV2 gene, in order to use sucrose as a single carbon source. deleting the INV2 gene was introduced into the INV2 gene on the vector by means a system for selecting a strain growing in a sucrose medium according to whether the expression of the INV2 gene.

The autoselection reporter gene of the present invention is not necessarily limited thereto, but is selected from the group consisting of invertase, sucrose, cellulose, xylase, maltease, amylase, glucoamylase, galactosidase, and the like. do.

In the present invention, "warm-expressing protein" refers to a protein that is difficult to produce recombinant expression in a host cell such as E. coli or yeast due to the characteristics of the protein itself when trying to recombinantly produce a protein derived from the human body or various organisms. For the purposes of the present invention, the poorly expressed protein may preferably comprise a large number of proteins that are not economically low in yeast, even though recombinant production is possible in other hosts such as E. coli.

The scope of application of the protein fusion factors TFP-1, TFP-2, TFP-3, TFP-4, TFP1-3, and TFP1-4 obtained in the present invention is human interleukin-2 (IL-2) and human colony stimulating factor ( Proteins, which are mass produced for a variety of commercial purposes, including G-CSF). Such 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, colonies Stimulating factor (GM-CSF), platelet induced growth factor (PDGF), phospholipase-activated protein (PLAP), insulin, tumor necrosis factor (TNF), growth factor (e.g., tissue growth factors such as TGFα or TGFβ and Endothelial growth factor), hormones (e.g. follicle-stimulating hormone, thyroid-stimulating hormone, antidiuretic hormone, pigment hormone and parathyroid hormone, progesterone-releasing hormone and its analogues), calcitonin, calcitonin gene related peptide (Calcitonin) Gene Related Peptide (CGPR), enkephalin, somatomedin, erythropoietin, hypothalamic secretion factor, prolactin, chronic gonadotropin, tissue plasminogen activator, growth hormone secretion peptide (gr owth hormone releasing peptide (GHPR) and thymic humoral factor (THF). Such proteins will also include enzymes, including 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.

The poorly expressed protein gene is a gene encoding the above-described proteins, which is selected or chemically synthesized from genomes or cDNAs derived from various animals and plants and microorganisms including the human body having human medical or industrial importance and the need for recombinant production.

The automatic selection vector of the present invention includes a promoter gene, a gene encoding a target protein from which translation start and stop codons have been removed, and a reporter gene fused in-frame thereto, and the promoter genes are GAPDH, PGK, ADH, PHO5 , GAL1, and GAL10. It is preferred that the gene is selected from the group consisting of a fusion factor library for discovering a custom fusion factor suitable for poorly expressed proteins include those that are selected or chemically synthesized from a genome or cDNA derived from a variety of flora and fauna, including the yeast or human body And preferably from yeast genes.

The host cell used for transformation in the smart selection method of the present invention is a Candida (Candida), di berry Oh, my process (Debaryomyces), Hanse Cronulla (Hansenula), Cluj Vero My process (Kluyveromyces), Pichia (Pichia), seukijo Saccharomyces My process (Schizosaccharomyces), Yarrow's (Yarrowia) and a saccharide as hyomoryu such as My process (Saccharomyces) genus Aspergillus (Aspergillus), Penny chamber William (Penicillium), rayijo Perth (Rhizopus), and Trichoderma (Trichoderma) in such Of fungi or bacteria such as Escherichia and Bacillus ( Bacillus ) genus may be used, but is not necessarily limited thereto.

The ultrafast screening method of tailor-made TFP for the production of refractory proteins of the present invention is suitable for use in non-expressable proteins that are impossible to express or have very low expression rates, but may be used to select TFPs which may increase the expression rate although the expression is somewhat high. Can be. As in the embodiment of the present invention, when invertase is used as a reporter, more efficient TFP can be selected by distinguishing them in the order of rapid growth in sucrose medium.

In another aspect, the present invention relates to a customized fusion factor ultrafast selection vectors pYHTS-F0, F1 and F2 for promoting the secretion production of the poor expression protein interleukin-2, and the selection vector is a protein of human expression interleukin-2 Butase contains a fused gene and a restriction enzyme Bam HI cleavage site made of three different reading frames at the amino terminus of the interleukin-2 gene.

In a specific embodiment of the present invention, the yeast chromosome is randomly cut and inserted into three selection vectors (pYHTS-F0, F1 and F2) for ultrafast selection of a customized fusion factor that promotes secretion of human interleukin-2 in yeast. By transforming the strains lacking invertase and selecting the cells growing in the sucrose medium, a customized fusion factor capable of secreting the fusion protein of the non-expressing interleukin-2 and invertase into the medium was selected.

Human interleukin-2 is a hydrophobic protein that is difficult to express in yeast due to the problem that the recombinant mass-expressed protein by a strong promoter does not fold rapidly into active form in the endoplasmic reticulum and aggregates with each other to paralyze the endoplasmic reticulum function. It is estimated. Thus, the invertase fused to interleukin-2 is also not secreted and cells cannot grow in sucrose medium. A protein fusion factor capable of efficiently secreting such a fusion protein is made possible by inserting a yeast genomic library at the front of the interleukin-2 gene, transforming it into yeast, and securing a transformant growing in sucrose medium.

In order to secure a fusion factor that induces the secretion of interleukin-2, an ovarian expression protein using the present invention, genes are separated from transformants growing in sucrose medium and retransformed into E. coli. pYHTS-TFP1, TFP2, TFP3 and TFP4) were recovered. Four different protein fusion factor genes TFP-1 (SEQ ID NO: 2), TFP-2 (SEQ ID NO: 4), TFP-3 (SEQ ID NO: 6), and TFP-4 (SEQ ID NO: 8) inserted into each plasmid The amino acid sequences encoded by these genes are shown in SEQ ID NOs: 1, 3, 5, and 7, respectively.

PYIL-TFP1, TFP2, TFP3 and TFP4 were prepared by removing the invertase from the obtained vectors pYHTS-TFP1, TFP2, TFP3 and TFP4 and inserting a translation termination codon into the interleukin-2 gene. Since the secretion of the interleukin-2 in the fused form, vectors pYIL-KRTFP1, KRTFP-2, KRTFP-3 and KRTFP-4 having the proteinase Kex2p recognition site were inserted to remove the protein fusion factor. Furthermore, by constructing the vector pYGCSF-TFP1 in which human colony stimulating factor (G-CSF) was fused with protein fusion factor TFP-1, it was demonstrated that TFP-1 is effective in the production of proteins other than human interleukin-2.

Thus, as another aspect, the present invention relates to a protein fusion factor TFP-1 protein, analogs or fragments thereof, set forth in SEQ ID NO: 1. The invention also relates to a gene encoding a protein fusion factor TFP-1 protein, analogues or fragments thereof, as set forth in SEQ ID NO: 1. Alternatively, the present invention is directed to the amino acid sequence set forth in SEQ ID NO: 1 or homologous thereof, preferably 75%, more preferably 85%, more preferably 90%, most preferably at least 95% homology. It relates to a protein fusion factor TFP-1 protein having an amino acid sequence having a. In addition, the present invention provides a DNA encoding the protein fusion factor TFP-1 protein as set forth in SEQ ID NO: 1 or its homology, preferably 75%, more preferably 85%, even more preferably 90%, most preferably Relates to a gene having a DNA sequence having at least 95% homology. Preferably, said gene is a gene of SEQ ID NO: 2. The present invention also relates to a recombinant vector comprising the gene described above. Preferably, the gene included in the recombinant vector is a gene of SEQ ID NO. Preferably, the recombinant vector is pYIL-TFP1, pYIL-KRTFP1 or pYGCSF-TFP1. The present invention also relates to a cell transformed with the recombinant vector described above. Preferably, the transformed cells are Escherichia coli DH5 @ / pYIL-KRTFP1 (KCTC 10544BP) transformed with pYIL-KRTFP1.

In another aspect, the invention relates to a protein fusion factor TFP-2 protein, analogs or fragments thereof, set forth in SEQ ID NO: 3. The invention also relates to a gene encoding a protein fusion factor TFP-2 protein, analogues thereof or fragments thereof as set forth in SEQ ID NO: 3. Alternatively, the present invention relates to an amino acid sequence represented by SEQ ID NO: 3 or an amino acid having homology, preferably 75%, more preferably 85%, even more preferably 90%, most preferably at least 95% homology thereof. Protein fusion factor TFP-2 protein having a sequence. In addition, the present invention provides a DNA sequence encoding the protein fusion factor TFP-2 protein as set forth in SEQ ID NO: 3, or homology thereof, preferably 75%, more preferably 85%, still more preferably 90%, most preferably Preferably a gene with a DNA sequence having at least 95% homology. Preferably, said gene is a gene of SEQ ID NO: 4. The present invention also relates to a recombinant vector comprising the gene described above. Preferably, the gene included in the recombinant vector is a gene of SEQ ID NO. Preferably, the recombinant vector is pYIL-TFP2 or pYIL-KRTFP2. The present invention also relates to a cell transformed with the recombinant vector described above. Preferably, the transformed cells are Escherichia coli DH5 @ / pYIL-KRTFP2 (KCTC 10545BP) transformed with pYIL-KRTFP2.

In another aspect, the present invention relates to a protein fusion factor TFP-3 protein, analogs or fragments thereof, set forth in SEQ ID NO: 5. The invention also relates to a gene encoding a protein fusion factor TFP-3 protein, analogues thereof or fragments thereof as set forth in SEQ ID NO: 5. Alternatively, the present invention relates to an amino acid sequence set forth in SEQ ID NO: 5 or to an homology thereof, preferably 75%, more preferably 85%, even more preferably 90%, most preferably at least 95% homology. Protein fusion factor TFP-3 protein having a sequence. In addition, the present invention provides a DNA sequence encoding a protein fusion factor TFP-3 protein as set forth in SEQ ID NO: 5 or a homology thereof, preferably 75%, more preferably 85%, even more preferably 90%, most preferably Preferably a gene with a DNA sequence having at least 95% homology. Preferably, said gene is a gene of SEQ ID NO: 6. The present invention also relates to a recombinant vector comprising the gene described above. Preferably, the gene included in the recombinant vector is a gene of SEQ ID NO. Preferably, the recombinant vector is pYIL-TFP3 or pYIL-KRTFP3. The present invention also relates to a cell transformed with the recombinant vector described above. Preferably, the transformed cells are Escherichia coli DH5 @ / pYIL-KRTFP3 (KCTC 10546BP) transformed with pYIL-KRTFP3.

In another aspect, the invention relates to a protein fusion factor TFP-4 protein, analogue or fragment thereof, as set forth in SEQ ID NO: 7. The invention also relates to a gene encoding a protein fusion factor TFP-4 protein, analogues thereof or fragments thereof as set forth in SEQ ID NO: 7. Alternatively, the present invention relates to an amino acid sequence set forth in SEQ ID NO: 7 or to an homology thereof, preferably 75%, more preferably 85%, even more preferably 90%, most preferably at least 95% homology. Protein fusion factor TFP-4 protein having a sequence. In addition, the present invention provides a DNA sequence encoding a protein fusion factor TFP-4 protein as set forth in SEQ ID NO: 7 or homology thereof, preferably 75%, more preferably 85%, still more preferably 90%, most preferably Preferably a gene with a DNA sequence having at least 95% homology. Preferably, said gene is the gene of SEQ ID NO: 8. The present invention also relates to a recombinant vector comprising the gene described above. Preferably, the gene included in the recombinant vector is a gene of SEQ ID NO: 8. Preferably, the recombinant vector is pYIL-TFP4 or pYIL-KRTFP4. The present invention also relates to a cell transformed with the recombinant vector described above. Preferably, the transformed cells are Escherichia coli DH5 @ / pYIL-KRTFP4 (KCTC 10547BP) transformed with pYIL-KRTFP4.

In the present invention, the term "analogue" used for the protein fusion factor protein or gene is a functional equivalent that shows the protein fusion factor activity by inducing secretion production of the non-expression protein when the protein fusion factor gene is fused with the gene of the non-expression protein. For protein fusion factor proteins, for example, substitution of amino acids having the same properties (e.g., substitution of hydrophobic amino acids with other hydrophobic amino acids, substitution of hydrophilic amino acids with other hydrophilic amino acids, basic amino acids with other basic amino acids) Substitution, acidic amino acid to other acidic amino acid), deletion of amino acid, insertion, or a combination thereof.

As used herein, the term “fragment” as used for a protein fusion protein or gene does not affect the secretion of the non-expressing protein even if some of the sequences of the protein fusion factor genes found through the genomic or cDNA library are removed Or a protein fusion factor gene or protein encoded by such a gene that promotes secretion.

The term "homologous" as used for the protein fusion factor protein or gene in the present invention is intended to indicate the degree of similarity between the wild type amino acid sequence and the wild type nucleic acid sequence, and in the case of a protein, the amino acid of the TFP protein of the present invention. It comprises an amino acid sequence which may preferably be at least 75%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% identical to the sequence. In general, a protein molar will comprise the same active site as the protein of interest. In the case of a gene, a DNA sequence which may be preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% identical to the DNA sequence encoding the TFP protein of the present invention. It includes. This homology comparison can be performed by using a comparison program that is easy to see with the naked eye. Commercially available computer programs can calculate the percent homology between two or more sequences, and the percent homology can be calculated for adjacent sequences.

The protein fusion factor found in accordance with the present invention for the production of secretory proteins of the non-expressive protein is used in fusion with the gene of the non-expressing protein and inserted into the vector for the secretion production of the non-expressing protein. In the present invention, a "vector" refers to a DNA sequence that is operably linked to a regulatory sequence capable of regulating the expression of a protein in a suitable host cell and sequences introduced to facilitate expression or to optimize the expression of the protein. DNA constructs containing. Such regulatory sequences include promoters to regulate transcription, operators optionally added to regulate transcription, appropriate mRNA ribosomal binding sites, and sequences to control termination of transcription / translation. As a vector for inserting a foreign gene, various types of vectors such as plasmids, viruses, and cosmids can be used. Vectors include cloning vectors and expression vectors, which are plasmids into which foreign DNA can be inserted and replicated, transferring foreign DNA to host bacterial cells during transformation. Expression vectors are usually carriers into which fragments of foreign DNA have been inserted and generally refer to fragments of double stranded DNA. Herein, foreign DNA refers to heterologous DNA, which is DNA not naturally found in host cells. Once in the host cell, the expression vector can replicate independently of the host chromosomal DNA and the inserted foreign DNA can be expressed. 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.

The term “transformation,” as used herein, with respect to transformation with a recombinant vector comprising a protein fusion factor, means that the DNA is introduced into a host such that the DNA is replicable as an extrachromosomal factor or by chromosomal integration. do. Host cells that can be used for transformation according to the present invention can include both prokaryotic or eukaryotic cells. A host having a high DNA introduction efficiency and a high expression efficiency of the introduced DNA is usually used. Bacteria, for example, known eukaryotic and prokaryotic hosts such as Escherichia, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera pruperferda (SF9), CHO, COS 1, Animal cells such as COS 7, BSC 1, BSC 40, BMT 10 and the like are examples of host cells that can be used. Preferably Escherichia coli can be used.

The present inventors investigated the effects of protein fusion factors TFP-1, TFP-2, TFP-3 and TFP-4 genes on the secretion of poorly expressed proteins, and for TFP-1, serine, alanine-rich sequence, N Vectors containing genes with the glycosylation site or both removed failed to secrete poorly expressed proteins. On the other hand, when the 5'-UTR (5'-untranslated region) was removed (pYIL-KRT1-4), the expression rate of the non-expressing protein increased more than three times, and when the additional sequence at the additional 3 'end was removed (pYIL). -KRT1-3) also induced the secretion of poorly expressed proteins.

In another aspect, the invention relates to a protein fusion factor TFP1-3 protein or analog thereof, set forth in SEQ ID NO: 9. The invention also relates to a gene encoding a protein fusion factor TFP1-3 protein or an analog thereof. Alternatively, the present invention relates to an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid having homology, preferably 75%, more preferably 85%, even more preferably 90%, most preferably at least 95% homology thereof. Protein fusion factor TFP1-3 protein having a sequence. In addition, the present invention provides a DNA sequence encoding the protein fusion factor TFP1-3 protein as set forth in SEQ ID NO: 9 or its homology, preferably 75%, more preferably 85%, still more preferably 90%, most preferably Preferably a gene with a DNA sequence having at least 95% homology. The present invention also relates to a recombinant vector comprising the gene described above. Preferably, the gene included in the recombinant vector is a gene encoding the protein fusion factor TFP1-3 described in SEQ ID NO: 9. Preferably, the recombinant vector is pYIL-KRT1-3. The present invention also relates to a cell transformed with the recombinant vector described above. Preferably, the transformed cells are Escherichia coli DH5 @ / pYIL-KRT1-3 (KCTC 10548BP) transformed with pYIL-KRT1-3.

In another aspect, the invention relates to a protein fusion factor TFP1-4 protein or analog thereof encoded by the gene set forth in SEQ ID NO: 10. The present invention also relates to a gene described by SEQ ID NO: 10 encoding a protein fusion factor TFP1-4 or an analog thereof. Alternatively, the present invention relates to amino acid sequences encoded by the genes set forth in SEQ ID NO: 10 or homologs thereof, preferably 75%, more preferably 85%, more preferably 90%, most preferably at least 95% It relates to a protein fusion factor TFP1-4 protein having an amino acid sequence having homology. In addition, the present invention provides a DNA sequence represented by SEQ ID NO: 10 or a homology thereof, preferably 75%, more preferably 85%, even more preferably 90%, most preferably a DNA having at least 95% homology. It relates to a gene having a sequence. The present invention also relates to a recombinant vector comprising a gene encoding a protein fusion factor TFP1-4 or an analog thereof. Alternatively, the present invention provides a DNA sequence encoding the protein fusion factor TFP-1-4, or its homology, preferably 75%, more preferably 85%, even more preferably 90%, most Preferably it relates to a recombinant vector comprising a gene having a DNA sequence having at least 95% homology. Preferably, the recombinant vector is pYIL-KRT1-4. The present invention also relates to a cell transformed with the recombinant vector described above. Preferably, the transformed cells are Escherichia coli DH5 @ / pYIL-KRT1-4 (KCTC 10549BP) transformed with pYIL-KRT1-4.

In another aspect, the invention provides a protein encoded by a protein as described in SEQ ID NO: 1, 3, 5, 7 or 9, an analog or fragment thereof, or a gene as described in SEQ ID NO: 10, an analog or fragment thereof The present invention relates to a method for producing a poorly expressed protein by a recombinant method. Alternatively, the present invention relates to an amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 7 or 9, or homology thereof, preferably 75%, more preferably 85%, even more preferably 90%, most preferably A protein having an amino acid sequence having at least 95% homology, or an amino acid sequence encoded by a gene set forth in SEQ ID NO: 10 or a homology thereof, preferably 75%, more preferably 85%, even more preferably 90 %, Most preferably a protein having an amino acid sequence having at least 95% homology, to a method for recombinantly producing a non-expressing protein. Preferably the protein described in SEQ ID NO: 1 is encoded by the gene described in SEQ ID NO: 2, the protein described in SEQ ID NO: 3 is encoded by the gene described in SEQ ID NO: 4, and the protein described in SEQ ID NO: Is encoded by the gene described in SEQ ID NO: 6, and the protein described in SEQ ID NO: 7 is encoded by the gene described in SEQ ID NO: 8. Preferably, the refractory protein is human interleukin-2 or human G-CSF.

Hereinafter, the present invention will be described in detail by way of examples. The following examples are merely illustrative of the present invention, but the content of the present invention is not limited to the following examples.

<Example 1> mutant strain lacking yeast invertase

The inventors of the present invention have prepared an automatic selection system using the yeast invertase as a reporter for ultrafast selection of protein fusion factors for poorly expressed proteins using cell growth in sucrose medium.

Specifically, to use the invertase gene included in the vector as a screening reporter gene after transformation, a yeast lacking invertase activity is required, and for this, the INV2 gene present in the chromosome is deleted. To prepare a gene deletion induction cassette, plasmid pRB58 (Carlson et al., Cell, 1982, 20, 145) was treated with restriction enzymes Eco RI and Xho I to recover the INV2 coding gene and introduced into the EcoRI / XhoI site of pBluescript KS +. Δ BX was prepared. As shown in FIG. 1, pBIU was prepared by inserting URA3 gene including 190 bp repetitive sequence (Tc190) at both ends into Hin dIII- Xba I site of INV2 gene included in pBI Δ BX. pBIU the restriction enzyme Eco RI- Xho I a saccharide in my process to the celebrity Y2805 △ gal1 busy then treated with (Mat a ura3 INV2 pep4 :: HIS3 gal1 can1) strain transformed with the transformant in selective medium without uracil conversion to body Y2805 Δgal1Δinv2U ( Mat a inv2 :: URA3 pep4 :: HIS3 gal1 can1) was selected.

To determine whether the invertase activity of the selected transformants was quenched, single colonies were cultured in each medium fed with glucose and sucrose as a single carbon source, and they grew normally in glucose and also in sucrose compared to the control. This was very slow but could grow. However, INV2 + strain and △ cultured inv2 strain, and then separating the proteins present in the culture supernatant by SDS-PAGE in sucrose solution can gel for 30 min in order to examine the amount of beote rise secreted into the culture medium, and then TTC (2,3,5-triphenyl-tetrazolium chloride ) results of a character aunt grams (zymogram) analysis with color development (Fig. 2) △ for inv2 strain was found that most of the lost invertase activity. However, there is a very slow but growing problem in sucrose medium, which is estimated to partially grow cells by gluconeogenesis through the function of mitochondria. Therefore, after removing antimycin A, a mitochondrial electron transport system inhibitor, to confirm the growth of the strain without expression of invertase, the growth of the strain could be completely suppressed (FIG. 3).

In order to retransform the URA3 vector containing the human cDNA library into the selected Y2805 Δgal1 △ inv2U ( Mat a inv2 :: URA3 pep4 :: HIS3 gal1 can1) strains, it is necessary to remove the URA3 gene used for deletion of the INV2 gene. To this end, strain Y2805 Δgal1Δinv2 ( Mat a ura3 inv2 :: Tc190 pep4 ), in which cultured cells were cultured in 5-fluoroorotic acid (5-FOA) medium and the URA3 gene was popped out. :: HIS3 gal1 can1) were selected (FIG. 1). Southern blotting confirmed whether the INV2 gene on the chromosome was deleted as expected and the URA3 gene was popped out again (FIG. 4). When the chromosome of Y2805 strain was treated with Eco RI and the INV2 gene was used as a probe, a fragment of about 4.3 kb was identified. After the URA3 gene was inserted (Y2805 Δgal1Δinv2U ), the URA3 gene was popped. If it is out (Y2805 ? Gal1 ? Inv2 ), it is reduced to about 3.7 kb. As shown in the results, the INV2 gene was correctly deleted as expected and the URA3 gene was popped out.

Example 2 Confirmation of Automatic Screening System through Fusion with Invertase

The beote rise gene deletion strain (Y2805 △ inv2) in the inverted table of human protein that is expressed in a yeast well to confirm that the automatic sorting of the sucrose medium through the expression of the fusion protein jeuwa human serum albumin (human serum albumin, HSA) and human expression protein interleukin-2 (IL-2).

First, the present inventors have used the Sfi I recognition having the sequence sense for a polymerase chain reaction primer JH97 (SEQ ID NO: 11) and antisense primer JH119 (SEQ ID NO: 12) to create a vector pGHSA-INV2 fused to the albumin and in beote Izu pYHSA5 (Kang et al., J. Microbiol. Biotechnol., 1998, 8, 42) as a template and polymerase chain reaction using Pfu polymerase (Stratazine, USA) (once at 94 ° C. for 5 minutes; 94 ° C.) The reaction was performed 25 times for 30 seconds at 55 ° C. for 30 seconds at 25 ° C. for 2 minutes; once at 72 ° C. for 7 minutes) to obtain an albumin gene of about 1.8 kb. In addition, using primers JH99 (SEQ ID NO: 13) and JH100 (SEQ ID NO: 14) with pRB58 as a template, the invertase gene was recovered by polymerase chain reaction in the same manner, and then treated with restriction enzyme Sfi I / Sal I and Pst. The albumin gene treated with I / Sfi I was inserted into pBluescript (Stratazin, USA) treated with Pst I / Sal I. pYHSA5 was treated with restriction enzyme Sac I / Pst I to obtain a fragment containing the GAL promoter and a portion of the albumin gene, and the Pst I / Sal I fragment containing the previously prepared albumin gene and the invertase gene was returned to Sac I / Sal. PGHSA-INV2 was prepared by simultaneous insertion into I treated YEGα-HIR525 vectors (Choi et al., Appl Microbiol Biotechnol., 1994, 42, 587). For the fusion expression vector, production of interleukin-2 and the interleukin-2 gene in beote rise primer JH106 (SEQ ID NO: 15) and JH107 by the pBKS-IL2 containing the gene obtained using (SEQ ID NO: 16) treated with Sfi I fragment and GAL promoter and inserted into the Sac I- Sfi I fragment containing the INV secretory signal at the same time the pGHSA-INV2 treated with Sac I / Sfi I was prepared pGIL2-INV2.

Vectors of pGHSA-INV2 expressing albumin-invertase fusion protein, pGIL2-INV2 expressing fusion protein of interleukin-2 and invertase, and pRB58 expressing only invertase were deleted from sucrose. were transformed respectively to not grow in the medium the strain (Y2805 △ inv2), to obtain a medium (UD) and the sucrose was added to the glucose as a carbon source plated on the addition of a carbon source medium (YPSA) observing the growth whether the cells (Fig. 5). In the case of pRB58, a vector that normally expresses invertase, both carbon sources grew well. Also, when invertase was fused with albumin, a protein that is well expressed in yeast (pGHSA-INV2), both carbon sources Grow well and use. However, in the case of interleukin-2, a protein that is difficult to express in yeast (pGIL2-INV2), it grew normally in glucose medium but not in sucrose medium. As expected, it was determined that interleukin-2 was not able to be expressed in the cell, and thus, the invertase fused thereto could not be expressed. Therefore, it was found that the automatic selection was possible by using the expression of the invertase gene transformed in the strain (Y2805 Δ inv2) that the invertase gene was deleted and could not be grown in the sucrose medium.

<Example 3> Preparation of protein fusion factor selection vector using the oocyte expression protein human interleukin-2

Library preparation vectors pYHTS-F0, F1 and three reading frames to obtain an appropriate protein fusion factor capable of inducing the secretion of the fusion protein to the vector pGIL2-INV2 fused with interleukin-2 and invertase. F2 was prepared (FIG. 6). Sense primers JH120 (SEQ ID NO: 17), JH121 (SEQ ID NO: 18), and JH122 (SEQ ID NO: 19) and antisense primer JH123 (SEQ ID NO: 20) having three reading frames and a Bam HI recognition sequence Polymerization chain reaction using PG polymerase (Stratazine, USA) using pGIL2-INV2 as a template (once for 3 minutes at 94 ° C; 30 minutes for 55 ° C, 30 seconds for 55 ° C, 30 seconds for 72 ° C 1 time at 72 ° C. for 7 min) to obtain 1.2 kb fragments each containing interleukin-2 and some invertase genes. Using a JH124 (SEQ ID NO: 21) and a JH95 (SEQ ID NO: 22) as a primer and pGIL2-INV2 as a template, the polymerase chain reaction was carried out under the same conditions to obtain a fragment of about 0.9 kb containing a part of the invertase gene. Respectively obtained. Each gene was recovered from the agarose gel and then mixed with three 1.2 kb fragments and 0.9 kb fragments with three reading frames, respectively, for the sense primers JH120 (SEQ ID NO: 17), JH121 (SEQ ID NO: 18), and JH122 (SEQ ID NO: 19) and the antisense primer JH95 (SEQ ID NO: 22) was carried out a secondary polymerase chain reaction and three kinds of 2.1 kb fragments were recovered by agarose gel electrophoresis. Three recovered 2.1kb fragments were treated with restriction enzymes Bam HI and Sal I to bind pGIL2-INV2 treated with Bam HI and Sal I to prepare pYHTS-F0, F1 and F2, respectively.

Example 4 Preparation of Custom Protein Fusion Factor Libraries from the Yeast Genome

The chromosomal DNA derived from yeast Saccharomyces cerevisiae and Hansenula polymorpha was used to prepare a protein fusion factor library. Each chromosome was partially cut with restriction enzyme Sau 3AI, recovered from 0.5 to 1.0 kb of DNA in agarose gel, digested with Bam HI, and treated with calf intestine phosphatase (pYHTS-F0). , F1 and F2 mixed vectors (FIG. 6). The linked DNA was transformed into Escherichia coli DH5α, plated in LB medium containing 1% Bacto-tryptone (1% Bacto-tryptone, 1% NaCl) and cultured at 37 ° C for one day. Transformant libraries of about 5 × 10 ⁴ cells were obtained using library DNA prepared from each chromosome. All transformants were recovered using sterile distilled water and library DNA was isolated from the recovered cells using a plasmid extraction kit (Bioneer).

Example 5 Automatic Selection of Customized Protein Fusion Factors Suitable for Human Secretory Protein Interleukin-2

The library DNA prepared by the above method was added to the yeast Saccharomyces cerevisiae in Y2805 Δgal1Δinv2 ( Mat a ura3 inv2 :: Tc190 pep4 :: HIS3 gal1 can1) and the lithium acetate method (Hills et al., Nucleic acids Res. 1991). , 19, 5791), followed by uracil-deficient UD minimal medium (a yeast substrate lacking 0.67% amino acid, a variety of amino acid mixtures at appropriate concentration, 2% glucose) and a complex medium containing sucrose and antimycin A YPGSA (1% yeast extract, 2% peptone, 2% sucrose, 0.03% galactose, 1 μg / mL antimycin A) was plated and incubated at 30 ° C. for 5 days. The number of cells formed in each medium after the culture is shown in Table 1 (comparison of the number of transformants before and after introduction of the protein fusion factor). When only the vectors (pYHTS-F0, F1, and F2) used for the production of the library were transformed, about 1 × 10 ⁴ cells were formed in glucose medium, but they did not grow at all in the medium using sucrose as a carbon source. I couldn't. However, when the yeast genome library was inserted, about 11 transformants were grown and the invertase was secreted with the help of the inserted protein fusion factor.

Example 6 Analysis of Protein Fusion Factor

Each cell grown in sucrose medium was incubated in YPD (1% yeast extract, 2% peptone, 2% glucose) for 24 hours, and then the recovered cells were disrupted, and then the introduced plasmids were isolated and retransformed into E. coli. After separating the plasmid from transformed Escherichia coli, restriction enzyme treatment was used to confirm the presence of the inserted gene, and sequencing analysis showed that the genes having four different sequences were inserted to act as protein fusion factors. Table 2: Novel protein fusion factors inserted into plasmids isolated from cells grown in sucrose medium).

Plasmid Convergence factor Yeast gene Number of fused amino acids (number of total amino acids) Characteristic pYHTS-TFP1pYHTS-TFP2pYHTS-TFP3pYHTS-TFP4 TFP-1TFP-2TFP-3TFP-4 Yar066wYar026cYjl158cUnknown 105 (203) 117 (169) 104 (227) 50 (unknown) PRE, N-gly, Ser-rich, GPIPRE, N-glyPRE-PRO, O-gly, PIRPRE

1.Translational fusion partner 1 (TFP-1)

 The inventors have discovered protein fusion factor 1 (TFP-1) (SEQ ID NO: 2) capable of efficiently secreting interleukin-2 and invertase fusion proteins out of cells. TFP-1 is the same gene as the yeast Saccharomyces cerevisiae gene Yar066w, similar to α1,4-glucan glucosidase (STA1), and glycosylphosphatidylinositol (GPI) -anchor ) Is a protein containing a gene whose function is not yet known. The number of amino acids fused with interleukin is 105 out of 203 and contains 23 amino acid secretion signals as signals for protein secretion, and contains N-glycosylation sites, serine and alanine-rich sequences. Doing.

2. Protein Fusion Factor 2 (TFP-2)

The present inventors discover interleukin-2 with a rise beote fusion protein effective to cell fusion protein factor 2 (TFP-2) that can be secreted out of the (SEQ ID NO: 4). TFP-2 is the same gene as the yeast Saccharomyces cerevisiae gene Yar026c and is yet unknown. The number of amino acids fused with interleukin-2 is 117 of 169 in total and contains a secretion signal of 19 amino acids as a signal for protein secretion, and contains three N-glycosylation sites.

3. Protein Fusion Factor 3 (TFP-3)

The inventors have discovered protein fusion factor 3 (TFP-3) (SEQ ID NO: 6) that can efficiently secrete interleukin-2 and invertase fusion proteins out of cells. TFP-3 is the same gene as the yeast Saccharomyces cerevisiae gene Yjl158c (CIS3), an O -mannosylated protein covalently bound to the cell wall, and a multicopy suppressor for Cik1 lacking strains involved in cell division. Is a gene known as). The number of amino acids fused with interleukin-2 is 104 out of 227 and contains 23 amino acid pre-secretion signals and 41 pro-secretion sequences as Lys-Arg. Contains the constructed Kex2p cleavage sequence and PIR repeat sequence.

4. Protein Fusion Factor 4 (TFP-4)

The inventors have discovered protein fusion factor 4 (TFP-4) (SEQ ID NO: 8) that can efficiently secrete interleukin-2 and invertase fusion proteins out of cells. TFP-4 is a gene derived from yeast Hanshenula polymorpha and its function is unknown, but there are 50 amino acids fused with interleukin-2, including a protein secretion signal consisting of 18 amino acids.

Example 7 Analysis of Fusion Proteins Secreted with Medium

To analyze the proteins secreted by the cells grown in sucrose medium, the cells containing the above four protein fusion factors were cultivated in YPDG medium (1% yeast extract, 2% peptone, 2% glucose, 0.03% galactose) for 40 hours. After incubation, the cells were removed, and the total protein dissolved in the remaining culture supernatant was precipitated with acetone (40% final concentration) and then subjected to SDS-PAGE. However, due to excessive glycosylation of the invertase part in the invertase fused state, Since there was a problem in identifying the protein band, the invertase was removed from each vector (pYHTS-TFP1, 2, 3, and 4) and a translation termination codon was inserted into the interleukin-2 gene. For this purpose primers JH132 (SEQ ID NO: 23) and JH137 (SEQ ID NO: 24) was used in the after each of the polymerase chain reaction process of the recovered gene fragment with Sac I / Sal I, treated with Sac I / Sal I YEGα-HIR525 PYIL-TFP1, 2, 3 and 4 were prepared by insertion into the vector. The four IL-2 expression vectors thus prepared were transformed into yeast, and a single colony was obtained and cultured in the same manner as described above, and the proteins secreted in the culture supernatant were analyzed by SDS-PAGE (FIG. 7). As shown in the results, strong bands with different protein sizes were observed in the culture supernatants of cells containing each vector except pYIL-TFP2, and they were identified as bands by Western blotting using IL-2 antibody (FIG. 7). As a result, each secretion-inducing fusion protein and IL-2 were in a fused state. However, there was a significant difference in the actual size shown on the SDS-PAGE than the protein size inferred from each protein fusion factor and IL-2 gene size, which was estimated to be due to the sugar chain addition, which was added to the protein by N -glycosylation. In order to remove sugars, each protein was treated with Endo-H enzyme and analyzed again by SDS-PAGE (FIG. 8). TFP-1 has one N -glycosylation inducing sequence (amino acids 28 to 30) in the protein sequence, TFP-3 contains an O -glycosylation inducing sequence, and TFP-4 has no sugar chain portion inducing sequence. As expected, in the case of pYIL-TFP1, the molecular weight was significantly reduced after the endoH treatment. Therefore, in the case of pYIL-TFP1, it was confirmed that the expressed protein was N -glycosylated. However, in the case of pYIL-TFP3, since the glycoside was formed by O -glycosylation, there was no change in molecular weight even after endoH treatment and pYIL-TFP4. There was no change in the case.

Example 8 Authentic Protein Production by Kex2p Cleavage in Cells

Since the vector used in Example 7 is secreted into the medium in the state where each protein fusion factor and IL-2 are fused together, the inventors used the protein in the cell to produce an authentic protein in the same state as human interleukin-2. In order to automatically remove the fusion factor, a cleavage site (Leu-Asp-Lys-Arg) recognized by the yeast protease Kex2p was inserted between the fusion factor and IL-2. To put the Kex2p cleavage sequence into pYIL-TFP1, pYIL-TFP1 was used as a template and polymerized using primers JH132 (SEQ ID NO: 23) and HY22 (SEQ ID NO: 25), HY23 (SEQ ID NO: 26), and JH137 (SEQ ID NO: 24), respectively. Enzyme chain reaction and the recovered two gene fragments as a template and the fragments obtained after the secondary polymerase chain reaction using JH132 (SEQ ID NO: 23) and JH137 (SEQ ID NO: 24) were treated with restriction enzyme Sac I / Sal I. And inserted into a YEGα-HIR525 vector treated with Sac I / Sal I to prepare pYIL-KRTFP1. In addition, pYIL-TFP2 was used as a template to insert Kex2p cleavage sequence into pYIL-TFP2, and primers JH132 (SEQ ID NO: 23) and HY20 (SEQ ID NO: 27), HY21 (SEQ ID NO: 28), and JH137 (SEQ ID NO: 24), respectively, were used. After the polymerase chain reaction, the recovered two gene fragments were used as templates, and the fragments obtained after the secondary polymerase chain reaction using JH132 (SEQ ID NO: 23) and JH137 (SEQ ID NO: 24) were used as restriction enzyme Sac I / Sal I. PYIL-KRTFP2 was prepared by insertion into a YEGα-HIR525 vector treated with Sac I / Sal I. In addition, pYIL-TFP3 was used as a template to insert Kex2p cleavage sequence into pYIL-TFP3, and primers JH132 (SEQ ID NO: 23) and HY17 (SEQ ID NO: 38), HY18 (SEQ ID NO: 39), and JH137 (SEQ ID NO: 24), respectively, were used. After the polymerase chain reaction, the recovered two gene fragments were used as templates, and the fragments obtained after the secondary polymerase chain reaction using JH132 (SEQ ID NO: 23) and JH137 (SEQ ID NO: 24) were used as restriction enzyme Sac I / Sal I. PYIL-KRTFP3 was prepared by insertion into a YEGα-HIR525 vector treated with Sac I / Sal I. In addition, in order to put the Kex2p cleavage sequence into pYIL-TFP4, pYIL-TFP4 was used as a template and primers JH132 (SEQ ID NO: 23), HY24 (SEQ ID NO: 29), HY25 (SEQ ID NO: 30), and JH137 (SEQ ID NO: 24) were used. After the polymerase chain reaction, the recovered two gene fragments were used as templates, and the fragments obtained after the secondary polymerase chain reaction using JH132 (SEQ ID NO: 23) and JH137 (SEQ ID NO: 24) were used as restriction enzyme Sac I / Sal I. PYIL-KRTFP4 was prepared by insertion into a YEGα-HIR525 vector treated with Sac I / Sal I.

Among the four vectors prepared, pYIL-KRTFP1, pYIL-KRTFP3 and pYIL-KRTFP4 were introduced into the yeast 2805 Δgal1Δinv2 strain, respectively, to select single colonies, and to select YPDG medium (1% yeast extract, 2% peptone, 2% glucose, 0.03% galactose) after 40 hours of removal of cells and the remaining culture supernatant SDS-PAGE, as shown in Figure 9 it was confirmed that the same size of the protein produced by human interleukin-2 secretion. Of the three protein fusion factors that produce human interleukin-2, it was confirmed that TFP-1 can most efficiently produce circular interleukin-2.

The vectors pYIL-KRTFP1, pYIL-KRTFP2, pYIL-KRTFP3, and pYIL-KRTFP4 were assigned to the Korean Collection for Type Cultures (KCTC), No. 52, Ueun-dong, Yuseong-gu, Daejeon, November 11, 2003, respectively. Deposited as 10545BP, 10546BP and 10547BP.

Example 9 Characterization of Protein Fusion Factor 1 (TFP-1)

As described above, TFP-1 is selected as a protein fusion factor that most efficiently produces IL-2, and thus, secretion signal (a), N -glycosylation site (b), and serine and alanine present in the TFP-1 sequence. To determine which of the rich sequence (c), the additional sequence (d) and the 5'-UTR (5'-untranslated sequence) sequence (e) has an absolute effect on the secretion of the secreted protein interleukin-2 As shown in FIG. 10, the missing gene was prepared by removing each feature sequence of TFP-1 one by one. First, pYIL-KRTFP1 was used as a template to remove the additional sequence d having no characteristic features in TFP-1, and polymerase chain reaction was performed using primers JH143 (SEQ ID NO: 31) and JH132 (SEQ ID NO: 23). The obtained DNA fragment contained a GAL promoter and TFP1-1, a fragment from which sequence d was removed from TFP-1. PYIL-KRTFP1 was used as a template to remove additional sequence d and serine, alanine-rich sequence (c) from TFP-1 and polymerase chain reaction using primers JH142 (SEQ ID NO: 32) and JH132 (SEQ ID NO: 23). The obtained fragment contains the GAL promoter and fragment TFP1-2 from which the sequences c and d have been removed. In addition, pYIL-KRTFP1 was used as a template to remove d, c and N-glycosylation site (b) from TFP-1 and polymerase chain reaction was carried out using primers JH141 (SEQ ID NO: 33) and JH132 (SEQ ID NO: 23). The obtained fragment contains the GAL promoter and the fragment from which the sequences c, d and b have been removed, including TFP1-3. To prepare the IL-2 gene including the Kex2p cleavage site, pYIL-KRTFP1 was used as a template and polymerase chain reaction was carried out using primers JH140 (SEQ ID NO: 34) and JH137 (SEQ ID NO: 24). The recovered IL-2 fragment was treated with restriction enzymes Spe I and Sal I and the three previously obtained fragments (TFP1-1, 2, and 3) were respectively treated with restriction enzymes Sac I and Xba I and Sac I and PYIL-KRT1-1, pYIL-KRT1-2, and pYIL-KRT1-3 were prepared as shown in FIG. 10 by insertion into YEGα-HIR525 treated with Sal I. To remove 5'-UTR of TFP-1, pYIL-KRTFP1 was used as a template, and polymerase chain reaction was carried out using primers HY38 (SEQ ID NO: 35) and JH137 (SEQ ID NO: 24), and the recovered gene was Bam HI / SAL I. PYIL-KRT1-4 was prepared by inserting into YEGα-HIR525 treated with Sac I / Sal I like the GAL10 promoter treated with Sac I / Bam HI (FIG. 10).

Four plasmids, pYIL-KRT1-1, pYIL-KRT1-2, pYIL-KRT1-3 and pYIL-KRT1-4, were transformed into yeast and cultured in a single colony. As shown in p. 11, pYIL-KRT1-3, which contains both secretion signals, N-glycosylation and serine, and alanine-rich sequences, only IL-2 bands were identified and pYIL- with serine and alanine-rich sequences removed. No bands were found in the case of pYIL-TFP1-1 from which KRT1-2 and serine, alanine-rich sequence and N-glycosylation sequence were removed. Therefore, in order to effectively induce IL-2 secretion, it can be seen that all three characteristic sequences present in TFP-1 (secretion signal, N-glycosylation and serine, alanine-rich sequence) are required. In addition, when the 5'-UTR of TFP1 was removed, the expression rate was confirmed to increase by more than three times.

The vectors pYIL-KRT1-3 and pYIL-KRT1-4 were deposited on November 11, 2003, under the accession numbers KCTC 10548BP and 10549BP, respectively, to KCTC (Korean Collection for Type Cultures), 52, Eeun-dong, Yuseong-gu, Daejeon, Korea. .

Example 10 Secretion Production of Human Colony Stimulating Factor (G-CSF) Using Protein Fusion Factor TFP-1

Protein fusion factor that can efficiently produce the human secreted interleukin-2, a secreted protein to determine whether TFP-1 is effective in the secretion of other human secreted protein, human colony stimulating factor (G-CSF) ) Was fused to TFP-1 to confirm expression and secretion in yeast. Human colony stimulator genes were obtained from the human cDNA library by polymerase chain reaction using primers JH144 (SEQ ID NO: 36) and JH145 (SEQ ID NO: 37). The obtained genes were treated with restriction enzyme Xba I / Sal I and pYIL. PYGCSF-TFP1 was prepared by insertion into the Xba I / Sal I site of -KRTFP1. As a control, MFα, a secretion signal derived from mating factor alpha, the secretion signal most commonly used in yeast, was used for the production of foreign protein secretion. A pYGCSF-MFα was prepared by inserting the G-CSF gene treated with Xba I / Sal I into the Xba I / Sal I site of YEGα-HIR525.

Two vectors pYGCSF-TFP1 and pYGCSF-MFα prepared to express human colony stimulating factor were transformed into yeast, single colonies were isolated and cultured, and the culture supernatant was subjected to SDS-PAGE and Western blotting using G-CSF antibody. The results are as shown in FIG. No G-CSF band was identified in the MFα secretion signal used as a control, but the G-CSF band was observed in the case of TFP-1. Western blotting using an antibody against G-CSF (Chemicon, USA) confirmed the expected band, indicating that G-CSF, a secretory protein, is effectively secreted by the protein fusion factor TFP-1. Therefore, TFP-1 was considered to be very useful as a protein fusion factor that can promote the secretion of various secretory proteins other than IL-2 or G-CSF.

The protein fusion factor obtained according to the present invention provides a technique for economically mass-producing various proteins that have not been recombinantly produced or have low expression rates.

1 is a diagram showing the deletion process of the invertase gene and pop-out process of the selection label.

2 is a zymogram for measuring invertase activity.

Lanes 1,2,3: wild type S .; S. cerevisiae 2805

Lane 4,5,6: the rise beote deletion mutant (S. cerevisiae 2805 △ inv2)

Figure 3 is a photograph showing the growth of the cells according to the carbon source.

INV2 : wild type S. Celebrity 2805

△ inv2 : Invertase deletion mutant strain ( S. cerevisiae 2805 △ inv2 )

4 shows Southern blotting results for confirming invertase gene deletion.

Lane 1,2: s. Celebrity Y2805 ura3 INV2,

Lanes 3,4: S. Celebrity Y2805 △ inv2U ( URA3 △ inv2 ),

Lane 5, 6: s. Celebrity Y2805 △ inv2 ( ura3 △ inv2 )

5 is a photograph confirming cell growth in glucose and sucrose medium.

Figure 6 is a diagram illustrating the manufacturing process of the plasmids pYHTS-F0, F1 and F2 and the yeast gene library.

7 shows the results of SDS-PAGE and Western blotting of the culture supernatant of cells containing four protein fusion factors.

Lane 1: size marker

Lane 2: Interleukin-2

Lane 3: culture supernatant cultured cells containing pYIL-TFP1

Lane 4: culture supernatant cultured cells containing pYIL-TFP2

Lane 5: culture supernatant cultured cells containing pYIL-TFP3

Lane 6: Culture supernatant cultured cells containing pYIL-TFP4

8 shows SDS-PAGE results before and after Endo-H treatment for sugar chain analysis.

Lane 1,-: Culture supernatant cultured with cells containing pYIL-TFP1, Endo-H.

Lane 1, +: Culture supernatant cultured with cells containing pYIL-TFP1, endocytosis treatment

Lane 2,-: Culture supernatant cultured with cells containing pYIL-TFP3, Endo-H.

Lane 2, +: culture supernatant cultured with cells containing pYIL-TFP3, endo-treated

Lane 3,-: Culture supernatant cultured with cells containing pYIL-TFP4, Endo-H.

Lane 3, +: culture supernatant cultured with cells containing pYIL-TFP4, endo-treated

Figure 9 shows the results of SDS-PAGE analysis of cell culture supernatant with or without Kex2p processing site.

Lane M: Size Indicator

Lane 1: culture supernatant of cells containing pYIL-TFP1

Lane 2: culture supernatant of cells containing pYIL-KRTFP1

Lane 3: culture supernatant of cells containing pYIL-TFP3

Lane 4: Culture supernatant of cells containing pYIL-KRTFP3

Lane 5: culture supernatant of cells containing pYIL-TFP4

Lane 6: Culture supernatant of cells containing pYIL-KRTFP4

10 is a schematic diagram showing plasmids prepared after gene deletion for the characterization of TFP-1.

11 shows SDS-PAGE results confirming interleukin-2 secretion by TFP-1-derived protein fusion factors (TFP1-1, 2, 3 and 4).

Lane M: Size Indicator

Lane S: Interleukin-2

Lane 1-1: Culture supernatant cultured cells containing pYIL-KRT1-1

Lane 1-2: Culture supernatant cultured cells containing pYIL-KRT1-2

Lane 1-3: Culture supernatant cultured cells containing pYIL-KRT1-3

Lane 1: culture supernatant cultured cells containing pYIL-KRTFP1

Lane 1-4: Culture supernatant cultured cells containing pYIL-KRT1-4

12 is SDS-PAGE and Western blotting results confirming the secretion of the secreted protein human colony stimulating factor (G-CSF) using the protein fusion factor TFP-1.

Lane M: Size Indicator

Lane 1: culture supernatant cultured cells containing pYIL-KRTFP1

Lane 2: culture supernatant cultured cells containing pYGCSF-TFP1

Lane 3: culture supernatant cultured cells containing pYGCSF-MFα

<110> Korea Research Institute of Bioscience and Biotechnology <120> Translational fusion partners for the secretory production of proteins <160> 39 <170> KopatentIn 1.71 <210> 1 <211> 105 <212> PRT <213> Saccharomyces cerevisiae <400> 1 Met Phe Asn Arg Phe Asn Lys Phe Gln Ala Ala Val Ala Leu Ala Leu 1 5 10 15 Leu Ser Arg Gly Ala Leu Gly Asp Ser Tyr Thr Asn Ser Thr Ser Ser 20 25 30 Ala Asp Leu Ser Ser Ile Thr Ser Val Ser Ser Ala Ser Ala Ser Ala 35 40 45 Thr Ala Ser Asp Ser Leu Ser Ser Ser Asp Gly Thr Val Tyr Leu Pro 50 55 60 Ser Thr Thr Ile Ser Gly Asp Leu Thr Val Thr Gly Lys Val Ile Ala 65 70 75 80 Thr Glu Ala Val Glu Val Ala Ala Gly Gly Lys Leu Thr Leu Leu Asp 85 90 95 Gly Glu Lys Tyr Val Phe Ser Ser Asp 100 105 <210> 2 <211> 430 <212> DNA <213> Saccharomyces cerevisiae <400> 2 gatcgtcata ttcactcttg ttctcataat agcagtccaa gttttcatct ttgcaagctt 60 tactatttct ttctttttat tggtaaactc tcgcccatta caaaaaaaaa agagatgttc 120 aatcgtttta acaaattcca agctgctgtc gctttggccc tactctctcg cggcgctctc 180 ggtgactctt acaccaatag cacctcctcc gcagacttga gttctatcac ttccgtctcg 240 tcagctagtg caagtgccac cgcttccgac tcactttctt ccagtgacgg taccgtttat 300 ttgccatcca caacaattag cggtgatctc acagttactg gtaaagtaat tgcaaccgag 360 gccgtggaag tcgctgccgg tggtaagttg actttacttg acggtgaaaa atacgtcttc 420 tcatctgatc 430 <210> 3 <211> 117 <212> PRT <213> Saccharomyces cerevisiae <400> 3 Met Thr Pro Tyr Ala Val Ala Ile Thr Val Ala Leu Leu Ile Val Thr 1 5 10 15 Val Ser Ala Leu Gln Val Asn Asn Ser Cys Val Ala Phe Pro Pro Ser 20 25 30 Asn Leu Arg Gly Lys Asn Gly Asp Gly Thr Asn Glu Gln Tyr Ala Thr 35 40 45 Ala Leu Leu Ser Ile Pro Trp Asn Gly Pro Pro Glu Ser Leu Arg Asp 50 55 60 Ile Asn Leu Ile Glu Leu Glu Pro Gln Val Ala Leu Tyr Leu Leu Glu 65 70 75 80 Asn Tyr Ile Asn His Tyr Tyr Asn Thr Thr Arg Asp Asn Lys Cys Pro 85 90 95 Asn Asn His Tyr Leu Met Gly Gly Gln Leu Gly Ser Ser Ser Asp Asn 100 105 110 Arg Ser Leu Asn Asp 115 <210> 4 <211> 424 <212> DNA <213> Saccharomyces cerevisiae <400> 4 gatctcattg gattcaagag aaagaaactc tatactggcg ccaaattagc agtgtcaaat 60 ttcgaaaagg tgatgacgcc ctatgcagta gcaattaccg tggccttact aattgtaaca 120 gtgagcgcac tccaggtcaa caattcatgt gtcgcttttc cgccatcaaa tctcagaggc 180 aaaaatggag acggtactaa tgaacagtat gcaactgcac tactttctat tccctggaat 240 ggacctcctg agtcattgag ggatattaat cttattgaac tcgaaccgca agttgcactc 300 tatttgctcg aaaattatat taaccattac tacaacacca caagagacaa taagtgccct 360 aataaccact acctaatggg agggcagttg ggtagctcat cggataatag gagtttgaac 420 gatc 424 <210> 5 <211> 104 <212> PRT <213> Saccharomyces cerevisiae <400> 5 Met Gln Phe Lys Asn Val Ala Leu Ala Ala Ser Val Ala Ala Leu Ser 5 9 14 19 Ala Thr Ala Ser Ala Glu Gly Tyr Thr Pro Gly Glu Pro Trp Ser Thr 24 29 34 Leu Thr Pro Thr Gly Ser Ile Ser Cys Gly Ala Ala Glu Tyr Thr Thr 39 44 49 Thr Phe Gly Ile Ala Val Gln Ala Ile Thr Ser Ser Lys Ala Lys Arg 54 59 64 Asp Val Ile Ser Gln Ile Gly Asp Gly Gln Val Gln Ala Thr Ser Ala 69 74 79 84 Ala Thr Ala Gln Ala Thr Asp Ser Gln Ala Gln Ala Thr Thr Thr Ala 89 94 99 Thr Pro Thr Ser Ser Glu Lys Ile 104 <210> 6 <211> 642 <212> DNA <213> Saccharomyces cerevisiae <400> 6 gatcccgcct agcccttcca gcttttcttt ttcccctttt gctacggtcg agacacggtc 60 gcccaaaaga aacgggtcag cgtgtactgc gccaaaaaaa ttcgcgccga tttaagctaa 120 acgtccacaa acaaaaacaa aaataagaaa taggttgaca gtgggtgaaa aattctcgaa 180 ggtttcatct ccaaacagtc agtatataag tattcgggaa agagagccaa tctatcttgt 240 ggtgggtcta tcttaacctt ctctttttgg cagtagtaat tgtaaatcaa gacacataaa 300 actatttcac tcgctaaact tacatctaaa atgcaattca aaaacgtcgc cctagctgcc 360 tccgttgctg ctctatccgc cactgcttct gctgaaggtt acactccagg tgaaccatgg 420 tccaccttaa ccccaaccgg ctccatctct tgtggtgctg ccgaatacac taccaccttt 480 ggtattgctg ttcaagctat tacctcttca aaagctaaga gagacgttat ctctcaaatt 540 ggtgacggtc aagtccaagc cacttctgct gctactgctc aagccaccga tagtcaagcc 600 caagctacta ctaccgctac cccaaccagc tccgaaaaga tc 642 <210> 7 <211> 50 <212> PRT <213> Hansenula polymorpha <400> 7 Met Arg Phe Ala Glu Phe Leu Val Val Phe Ala Thr Leu Gly Gly Gly 5 9 14 19 Met Ala Ala Pro Val Glu Ser Leu Ala Gly Thr Gln Arg Tyr Leu Val 24 29 34 Gln Met Lys Glu Arg Phe Thr Thr Glu Lys Leu Cys Ala Leu Asp Asp 39 44 49 Lys yle 54 <210> 8 <211> 179 <212> DNA <213> Hansenula polymorpha <400> 8 gatccgcttt ttattgcttt gctttgctaa tgagatttgc agaattcttg gtggtatttg 60 ccacgttagg cggggggatg gctgcaccgg ttgagtctct ggccgggacc caacggtatc 120 tggtgcaaat gaaggagcgg ttcaccacag agaagctgtg tgctttggac gacaagatc 179 <210> 9 <211> 71 <212> PRT <213> Saccharomyces cerevisiae <400> 9 Met Phe Asn Arg Phe Asn Lys Phe Gln Ala Ala Val Ala Leu Ala Leu 1 5 10 15 Leu Ser Arg Gly Ala Leu Gly Asp Ser Tyr Thr Asn Ser Thr Ser Ser 20 25 30 Ala Asp Leu Ser Ser Ile Thr Ser Val Ser Ser Ala Ser Ala Ser Ala 35 40 45 Thr Ala Ser Asp Ser Leu Ser Ser Ser Asp Gly Thr Val Tyr Leu Pro 50 55 60 Ser Thr Thr Ile Ser Gly Asp 65 70 <210> 10 <211> 329 <212> DNA <213> Saccharomyces cerevisiae <400> 10 ggatccatgt tcaatcgttt taacaaattc caagctgctg tcgctttggc cctactctct 60 cgcggcgctc tcggtgactc ttacaccaat agcacctcct ccgcagactt gagttctatc 120 acttccgtct cgtcagctag tgcaagtgcc accgcttccg actcactttc ttccagtgac 180 ggtaccgttt atttgccatc cacaacaatt agcggtgatc tcacagttac tggtaaagta 240 attgcaaccg aggccgtgga agtcgctgcc ggtggtaagt tgactttact tgacggtgaa 300 aaatacgtct tctcatctga tcctctaga 329 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> JH97 primer <400> 11 ccggccatta cggccgtgat gcacacaaga gtgag 35 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> JH119 primer <400> 12 ccggccgagg cggcctaagc ctaaggcag 29 <210> 13 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> JH99 primer <400> 13 gggcggccgc ctcggcccta gataaaaggt caatgacaaa cgaaactagc 50 <210> 14 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> JH100 primer <400> 14 ccgtcgactt actattttac ttcccttact tg 32 <210> 15 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> JH106 primer <400> 15 gcggccatta cggccgtgca cctacttcaa gttctac 37 <210> 16 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> JH107 primer <400> 16 gcggccatta cggccgtgca cctacttcaa gttctac 37 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> JH120 primer <400> 17 cgggatccgc acctacttca agttct 26 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> JH121 primer <400> 18 cgggatcctg cacctacttc aagttct 27 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> JH122 primer <400> 19 cgggatcctt gcacctactt caagttct 28 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> JH123 primer <400> 20 ccattgaagg aaccaacaaa at 22 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> JH124 primer <400> 21 attttgttgg ttccttcaat gg 22 <210> 22 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> JH95 primer <400> 22 ggctcgagct attttacttc ccttacttg 29 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> JH132 primer <400> 23 gggagctcat cgcttcgctg att 23 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> JH137 primer <400> 24 ccgtcgactt aagttagtgt tgagatg 27 <210> 25 <211> 47 <212> DNA <213> Artificial Sequence <220> 223 HY22 primer <400> 25 gaacttgaag taggtgccct tttatctaga ggatcagatg agaagac 47 <210> 26 <211> 46 <212> DNA <213> Artificial Sequence <220> 223 HY23 primer <400> 26 tcttctcatc tgatcctcta gataaaaggg cacctacttc aagttc 46 <210> 27 <211> 46 <212> DNA <213> Artificial Sequence <220> 223 HY20 primer <400> 27 gaacttgaag taggtgccct tttatctaga ggatcgttca aactcc 46 <210> 28 <211> 46 <212> DNA <213> Artificial Sequence <220> 223 HY21 primer <400> 28 ggagtttgaa cgatcctcta gataaaaggg cacctacttc aagttc 46 <210> 29 <211> 47 <212> DNA <213> Artificial Sequence <220> 223 HY24 primer <400> 29 gaacttgaag taggtgccct tttatcaagg atcttgtcgt ccaaagc 47 <210> 30 <211> 47 <212> DNA <213> Artificial Sequence <220> 223 HY25 primer <400> 30 gctttggacg acaagatcct tgataaaagg gcacctactt caagttc 47 <210> 31 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> JH143 primer <400> 31 cctctagaat caccgctaat tgttgtg 27 <210> 32 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> JH142 primer <400> 32 cctctagagg tgctattggt gtaagag 27 <210> 33 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> JH141 primer <400> 33 cctctagaac cgagagcgcc gcgagag 27 <210> 34 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> JH140 primer <400> 34 ggactagtct agataaaagg gcacc 25 <210> 35 <211> 42 <212> DNA <213> Artificial Sequence <220> 223 HY38 primer <400> 35 gaatttttga aaattcaagg atccatgttc aatcgtttta ac 42 <210> 36 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> JH144 primer <400> 36 cctctagata aaaggacccc cctgggccct gcc 33 <210> 37 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> JH145 primer <400> 37 ggcagctgga tgtattttac atggggag 28 <210> 38 <211> 43 <212> DNA <213> Artificial Sequence <220> 223 HY17 primer <400> 38 gaacttgaag taggtgccct tttatcaagg atcttttcgg agc 43 <210> 39 <211> 43 <212> DNA <213> Artificial Sequence <220> 223 HY18 primer <400> 39 gctccgaaaa gatccttgat aaaagggcac ctacttcaag ttc 43

Claims (16)

Protein Fusion Factor TFP Proteins Selected From: (a) a protein fusion factor TFP-1 protein having an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90% homology thereto and exhibiting protein fusion factor TFP-1 protein activity; (b) a protein fusion factor TFP-2 protein having an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90% homology thereto and exhibiting protein fusion factor TFP-2 protein activity; (c) a protein fusion factor TFP-3 protein having an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90% homology thereto and exhibiting protein fusion factor TFP-3 protein activity; (d) a protein fusion factor TFP-4 protein having an amino acid sequence as set forth in SEQ ID NO: 7 or an amino acid sequence having at least 90% homology thereto and exhibiting protein fusion factor TFP-4 protein activity; (e) a protein fusion factor TFP1-3 protein having an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence having at least 90% homology thereto and exhibiting protein fusion factor TFP1-3 protein activity; And (f) a protein fusion factor TFP1-4 protein having an amino acid sequence encoded by the gene set forth in SEQ ID NO: 10 or an amino acid sequence having at least 90% homology thereto and exhibiting protein fusion factor TFP1-4 protein activity. Protein fusion factor TFP gene selected from: (a) a DNA encoding a protein fusion factor TFP-1 protein as set forth in SEQ ID NO: 1 or a DNA encoding a protein fusion factor TFP-1 protein activity having at least 90% homology thereto; (b) a DNA encoding a protein fusion factor TFP-2 protein as set forth in SEQ ID NO: 3 or a DNA encoding a protein having a protein fusion factor TFP-2 protein activity having at least 90% homology thereto; (c) a DNA encoding a protein fusion factor TFP-3 protein as set forth in SEQ ID NO: 5 or a DNA encoding a protein fusion factor TFP-3 protein activity having at least 90% homology thereto; (d) a DNA encoding a protein fusion factor TFP-4 protein as set forth in SEQ ID NO: 7 or a DNA encoding a protein fusion factor TFP-4 protein activity having at least 90% homology thereto; (e) a DNA encoding a protein fusion factor TFP1-3 protein as set forth in SEQ ID NO: 9 or a DNA encoding a protein fusion factor TFP1-3 protein activity having at least 90% homology thereto; And (f) a DNA as set forth in SEQ ID NO: 10 or a DNA encoding a protein fusion factor TFP1-4 protein activity having at least 90% homology therewith. The protein fusion factor TFP gene according to claim 2, wherein the protein fusion factor TFP gene is selected from the DNAs of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8. Recombinant vector comprising a gene selected from the DNA of claim 2 (a) to (f). The recombinant vector of claim 4, wherein the recombinant vector comprises a gene selected from among SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10. 6. The method of claim 4, wherein pYIL-TFP1, pYIL-KRTFP1, pYGCSF-TFP1, pYIL-TFP2, pYIL-KRTFP2, pYIL-TFP3, pYIL-KRTFP3, pYIL-TFP4, pYIL-KRTFP4, pYIL-KRT1-3 and pYIL-KRT1-3 Vector selected from KRT1-4. A host cell transformed with the recombinant vector of claim 4. 8. Escherichia coli KCTC 10544BP transformed with pYIL-KRTFP1. The Escherichia coli KCTC 10545BP of claim 7 transformed with pYIL-KRTFP2. 8. Escherichia coli KCTC 10546BP transformed with pYIL-KRTFP3. The Escherichia coli KCTC 10547BP of claim 7 transformed with pYIL-KRTFP4. The Escherichia coli KCTC 10548BP of claim 7 transformed with pYIL-KRT1-3. The Escherichia coli KCTC 10549BP of claim 7 transformed with pYIL-KRT1-4. A protein fusion factor TFP protein having the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 7 or 9 or an amino acid sequence having at least 90% homology with the protein fusion factor TFP protein activity or the gene described as SEQ ID NO: 10 A method for producing a poorly expressed protein using a protein fusion factor TFP protein having an amino acid sequence encoded by or having an amino acid sequence having at least 90% homology thereto and exhibiting protein fusion factor TFP protein activity. The method of claim 14, wherein the poorly expressed protein is human interleukin-2 (IL-2) or human colony stimulating factor (G-CSF). The method of claim 14, wherein the protein described in SEQ ID NO: 1 is encoded by the gene described in SEQ ID NO: 2, and the protein described in SEQ ID NO: 3 is encoded by the gene described in SEQ ID NO: 4, The protein described is encoded by the gene described in SEQ ID NO: 6, and the protein described in SEQ ID NO: 7 is encoded by the gene described in SEQ ID NO: 8.