KR20120140577A - A novel fabricated translational fusion partner for secretory production of foreign protein, a process for preparing the foreign protein using the same and a screening method of the same - Google Patents

A novel fabricated translational fusion partner for secretory production of foreign protein, a process for preparing the foreign protein using the same and a screening method of the same Download PDF

Info

Publication number
KR20120140577A
KR20120140577A KR1020110060407A KR20110060407A KR20120140577A KR 20120140577 A KR20120140577 A KR 20120140577A KR 1020110060407 A KR1020110060407 A KR 1020110060407A KR 20110060407 A KR20110060407 A KR 20110060407A KR 20120140577 A KR20120140577 A KR 20120140577A
Authority
KR
South Korea
Prior art keywords
ala
ser
protein
pro
thr
Prior art date
Application number
KR1020110060407A
Other languages
Korean (ko)
Other versions
KR101653809B1 (en
Inventor
손정훈
박순호
배정훈
성봉현
이승구
김현진
임광묵
Original Assignee
한국생명공학연구원
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 한국생명공학연구원 filed Critical 한국생명공학연구원
Priority to KR1020110060407A priority Critical patent/KR101653809B1/en
Publication of KR20120140577A publication Critical patent/KR20120140577A/en
Application granted granted Critical
Publication of KR101653809B1 publication Critical patent/KR101653809B1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormones [GH] (Somatotropin)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts

Abstract

PURPOSE: An artificial protein translational fusion partner comprising the combination of pre-secretion signal and pro-secretion signal for producing and secreting foreign protein is provided to mass-produce foreign protein hardly mass-produced in yeast due to a traditional recombinant technique with hGH. CONSTITUTION: An artificial protein translational fusion partner for producing and secreting foreign protein comprises the pre-secreting signal and pro-secretion signal of protein translational fusion partner from yeast. The pre-secretion signal and pro-secretion signal are picked from different protein translational fusion partners. Pre-secretion signal is the protein in the sequence number from 1 (SEQ ID NO:1) to 25 (SEQ ID NO:25) the sequence number from 51 (SEQ ID NO:51) to 75 (SEQ ID NO:75]. Pro-secretion signal is the protein chosen in the sequence number from 1 (SEQ ID NO:1) to 25 (SEQ ID NO:25] or the gene chosen in the sequence number from 51 (SEQ ID NO:51) to 75(SEQ ID NO:75). The foreign protein is human growth hormone and pre-secretion signal is TFP 20-pre(the sequence number 14 and 64) and pro-secretion signal is TFP3-pre(the sequence number 3 and 53) or TFP 3-pre(the sequence number 10 and 60). A method of producing foreign protein comprises the following steps. An expression vector with an artificial protein translational fusion factor and a foreign protein gene to be produced is made(i). The expression vector in step (i) is introduced in yeast so that a transformant is produced(ii). The transformant is cultivated and foreign protein is obtained in a culture or a supernatant layer(iii).

Description

외래단백질 분비생산용 인공 단백질 분비융합인자, 이를 사용한 외래단백질의 생산방법 및 인공 단백질 분비융합인자의 스크리닝 방법{A novel fabricated translational fusion partner for secretory production of foreign protein, a process for preparing the foreign protein using the same and a screening method of the same}Artificial protein secretion fusion factor for the production of foreign protein secretion, production method of foreign protein using the same and screening method of artificial protein secretion fusion factor {A novel fabricated translational fusion partner for secretory production of foreign protein, a process for preparing the foreign protein using the same and a screening method of the same}

본 발명은 외래단백질 분비생산용 인공 단백질 분비융합인자, 이를 사용한 외래단백질의 생산방법 및 인공 단백질 분비융합인자의 스크리닝 방법에 관한 것으로, 보다 구체적으로 본 발명은 외래단백질을 분비생산하기 위한, 프리-분비시그널과 프로-분비시그널의 신규한 조합을 가지는 인공 단백질 분비융합인자(TFP), 상기 인공 단백질 분비융합인자를 이용하여 외래단백질을 분비생산하는 방법 및 인공 단백질 분비융합인자 라이브러리로부터 목적하는 외래단백질의 분비생산 효율이 가장 우수한 프리-분비시그널 및 프로-분비시그널의 조합으로 구성된 인공 단백질 분비융합인자를 스크리닝하는 방법에 관한 것이다.
The present invention relates to an artificial protein secretion fusion factor for foreign protein secretion production, a method for producing an exogenous protein using the same and a method for screening an artificial protein secretion fusion factor, and more specifically, the present invention for the production of secreted foreign protein, pre- Artificial protein secretion fusion factor (TFP) having a novel combination of secretion signal and pro-secretion signal, a method for secreting the foreign protein using the artificial protein secretion fusion factor and the foreign protein of interest from the artificial protein secretion fusion factor library The present invention relates to a method for screening an artificial protein secretion fusion factor consisting of a combination of pre-secretion signal and pro-secretion signal having the best secretion efficiency.

인간 성장호르몬(human Growth Hormone, hGH)은 뇌하수체전엽 호르몬 중 가장 많이 분비되는 단일쇄(single chain)의 단백질 호르몬으로서, 191개의 아미노산 잔기로 구성되는 분자량 약 22kDa의 호르몬이다. 성장호르몬은 뇌와 눈을 제외한 거의 모든 체내 조직 장기의 세포를 증식시켜 균형잡힌 성장을 촉진하며, 골성장으로 키가 커지고 골격근이 비대해지는 생리학적 작용을 가지고 있어 임상적으로는 뇌하수체 기능 부전성 왜소증 치료에 사용되고 있다. 이전에는 주로 교통사고나 질병으로 사망한 인간의 뇌하수체로부터 인간 성장호르몬을 추출 정제하여 사용하여 왔으나, 그 양이 크게 제한되어 있고, 또한 고도로 정제된 사람의 뇌하수체 호르몬만을 사용하여야하는 점 때문에 모든 왜소증 환자에게 공급되지 못하였으며, 가격 또한 높아 사용에 많은 어려움이 있었다. 더욱이, 추출 정제한 인간 성장호르몬을 투여받은 어린이가 괴질의 바이러스에 감염되어 사망한 사고가 발생하는 등, 그 사용에 문제점이 있어 미국 식품 의약국(FAD)에서는 사망한 인간의 뇌하수체로부터 추출정제한 인간 성장호르몬의 사용을 금지시켰다. 추출 이외에도 화학적 합성이나 조직 배양 방법 등도 있으나, 이들 역시 수율이 낮은 단점을 가지고 있어 생산 방법의 개선이 제기되어 왔다.Human Growth Hormone (hGH) is the most secreted single-chain protein hormone of the anterior pituitary hormone, a hormone of about 22 kDa consisting of 191 amino acid residues. Growth hormone promotes balanced growth by proliferating cells of almost all tissues in the body except brain and eyes, and has physiological effects that increase bone growth and enlarge skeletal muscle. Clinically, pituitary insufficiency dwarfism It is used for treatment. Previously, human growth hormone was extracted and used mainly from the human pituitary gland, which died of a traffic accident or disease. However, the amount of the human pituitary gland is limited and the use of highly purified human pituitary hormones all causes dwarfism patients. It was not supplied to the company, and the price was also high, so there were many difficulties in use. Furthermore, the US Food and Drug Administration (FAD) restricts the extraction of the dead human pituitary gland. The use of human growth hormone was banned. In addition to extraction, there are chemical synthesis and tissue culture methods, but these also have disadvantages of low yields, and thus improvement of production methods has been proposed.

최근 인체 게놈 프로젝트에서 확보된 유전체 염기서열 정보와 유전체 단위에서 밝혀지는 다양한 단백질의 기능을 분석하고 인체 의약학적으로 중요한 단백질 제품생산을 위해서는 재조합 미생물을 이용하는 고효율 단백질 생산 시스템 개발이 필요하다. 인체와 같은 고등생물 유래의 재조합단백질을 생산하기 위해서 발현시스템을 선정할 때 숙주세포의 성장특성, 단백질 발현정도, 세포내외 발현가능성, 번역 후 수식(post-translational modification) 가능성, 발현된 단백질의 생물학적활성 및 발현단백질의 용도 등과 같은 다양한 요인들이 고려되어야 한다. 대표적 미생물 유전자 발현시스템으로 에셔리키아 콜라이 및 효모 시스템이 주로 이용되고 있는데 에셔리키아 콜라이는 많은 발현시스템이 개발되어 있고 외래단백질의 발현율이 매우 높은 장점이 있지만 고등생물 유래의 단백질을 재조합 생산하고자 할 때 번역 후 수식 과정이 불가능하며 세포의 배양배지로 단백질의 완전한 분비가 어렵고 이황화 결합(disulfide bond)이 많은 단백질의 폴딩(folding)이 불가능하며 봉합체(inclusion body) 등의 불용성 단백질 형태로 생산하는 등의 단점이 지적되고 있다 (Makrides, MicrobialRev., 1996, 60, 512). 또한, 인체단백질 중 질병과 연관되어 의약학적으로 가치가 높은 대부분의 단백질이 당단백질이거나 막단백질이기 때문에 완전한 활성을 갖기 위해서는 글리코실화(glycosylation)를 반드시 요구하거나 이황화 결합을 통한 완전한 3차원 구조를 요구할 경우 에셔리키아 콜라이에서는 생산이 불가능하며 효모와 같은 진핵 미생물 발현 시스템을 반드시 필요로 한다.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 human pharmacologically important protein products, it is necessary to develop a highly efficient protein production system using recombinant microorganisms. 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 the representative microbial gene expression system, Escherichia coli and yeast system are mainly used. However, Escherichia coli has many expression systems and high expression rate of foreign protein. When post-translational modification is impossible, it is difficult to completely secrete protein by cell culture medium, and it is impossible to fold protein with many disulfide bonds and produce insoluble protein form such as inclusion body. And other drawbacks (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. Production in Escherichia coli is not possible and necessitates a eukaryotic microbial expression system such as yeast.

진핵 미생물인 효모 사카로마이세스 세레비시애(Saccharomyces cerevisiae)는 인체에 대해 안전성이 입증된 GRAS(Generally Recognized As Safe) 미생물로서 유전자 조작이 용이하며 다양한 발현시스템이 개발되어 있고 대량배양이 용이하다. 뿐만 아니라 인체단백질과 같은 고등세포 유래의 단백질을 재조합 생산할 때 단백질을 세포 밖으로 분비할 수 있는 분비기능과 글리코실화 등과 같은 단백질의 번역 후 수식 기능을 수행할 수 있는 장점을 제공한다. 단백질의 분비 시그날과 목표 단백질을 인위적으로 융합(fusion)함으로써 세포외 분비가 가능한데 단백질의 분비과정을 통해서 단백질의 폴딩이나 이황화 결합의 형성 및 당쇄부가 과정이 진행되며 따라서 생물학적으로 완전한 활성을 갖는 재조합 단백질을 생산할 수 있는 장점을 제공한다. 이는 또한 생물학적 활성을 갖는 단백질을 배지로부터 직접 얻을 수 있기 때문에 경제적으로 효율이 낮은 세포의 분쇄나 재접힘 단계를 필요로 하지 않아 매우 경제적이다 (Eckart and Bussineau, Curr. Opin.Biotechnol., 1996, 7, 525).The eukaryotic microorganism yeast Saccharomyces cerevisiae is a GRAS (Generally Recognized As Safe) microorganism that has been proven to be safe for humans, and is easily genetically engineered, various expression systems are developed, and mass culture is easy. In addition, when recombinant production of higher cell-derived proteins such as human proteins, it provides a secretion function to secrete the protein out of the cell and post-translational modification of the protein such as glycosylation. Extracellular secretion is possible by artificially fusion of the secretion signal of the protein with the target protein. The process of folding the protein, forming disulfide bonds, and adding sugar chains through the secretion process of the protein results in 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).

그러나 상기한 많은 장점에도 불구하고 효모 사카로마이세스 세레비시애를 이용한 인체 단백질 분비시스템에 관한 현 기술의 문제점으로 인체 단백질의 종류에 따라서 전혀 생산되지 않거나 수 그램/리터까지 생산되는 등 분비율이 수천 배 이상 차이를 보여 분비생산성을 예측하기 힘든 것이다. 외래단백질이 수 그램/리터 수준까지 분비 생산이 가능한 것으로 판단할 때 분비 생산성 측면에서 충분한 경제성이 있는 것으로 판단되나 단백질의 종류에 따라서 분비효율이낮은 문제와 특히 고부가가치의 인체 의약용 단백질을 생산하고자 할 때 발현 및 분비가 힘든 문제가 자주 발생한다. 따라서, 효모에서 인체 단백질을 대량생산하기 위해서는 단백질의 분비발현을 향상시키고 분비된 단백질의 엉킴을 막음으로써 완전한 형태의 인체 단백질의 생산방법의 개발이 필요하다.
However, despite many of the advantages described above, there is a problem of the current technology related to the human protein secretion system using 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 the secretion productivity by more than thousands of times. When exogenous protein can produce secretion up to several grams / liter, it seems that there is sufficient economic efficiency in terms of secretion productivity.However, it is necessary to produce low-efficiency problems and especially high-value human pharmaceutical protein depending on the type of protein. It is often difficult to express and secrete. Therefore, in order to mass-produce human proteins in yeast, it is necessary to develop a method for producing a human protein in a full form by improving secretion of proteins and preventing entanglement of secreted proteins.

이러한 문제를 해결하기 위해, 재조합 단백질의 분비에 관여하는 분비융합인자에 대한 연구가 많이 진행되고 있다. 그러나 개발된 분비 인자들이 특정단백질의 분비증진에는 효과가 있으나 모든 단백질에 일률적으로 적용될 수 없는 문제가 있다. 즉, 단백질 분비융합인자를 이용한 분비유도의 경우에도 단백질의 종류에 따라서 분비에 미치는 영향이 서로 상이했기 때문에 개발된 분비융합인자가 모든 단백질에 적용될 수 없는 문제가 있다. 따라서 목적하는 각 단백질 분비증진 최대화를 위해서는 목적 단백질에 특이적으로 적용될 수 있는 최적의 단백질 분비융합인자를 선별하는 기술 또한 필요하다. In order to solve this problem, a lot of researches on the secretion fusion factor involved in the secretion of recombinant proteins. However, the developed secretion factors are effective in promoting the secretion of specific proteins, but there is a problem that cannot be applied uniformly to all proteins. That is, even in the case of secretion induction using protein secretion fusion factor, there is a problem that the developed secretion fusion factor cannot be applied to all proteins because the effects on secretion are different from each other according to the type of protein. Therefore, in order to maximize each protein secretion desired, a technique for selecting an optimal protein secretion fusion factor that can be specifically applied to the target protein is also required.

이러한 배경하에, 본 발명자들은 효모에서 외래단백질을 고효율 분비생산하는 방법으로 효모 유전체로부터 다양한 분비단백질 유전자를 확보하고 외래단백질 맞춤형 분비융합인자 TFP(translational fusion partner) 기술을 개발한 바 있다(한국특허 제0626753 호, 한국특허 제0798894호, 한국특허 제0975596호). 발굴된 모든 TFP는 기능적으로 아미노말단에 20여 개의 아미노산으로 구성된 프리-분비시그널(pre-secretion signal)과 그에 연결된 프로-분비시그널(pro-secretion signal) 또는 분비융합파트너(secretion fusion partner)로 구성되어 있다. 그러나, 상기 각각의 TFP는 분비생산을 촉진시킬 수 있는 단백질이 제한되어 있어, 다양한 외래단백질의 분비생산에 적용하기가 어렵다는 단점이 있었다.
Under these circumstances, the present inventors have secured various secretory protein genes from the yeast genome and developed a foreign-tailored secretion fusion factor TFP (translational fusion partner) technology as a method of producing high-efficiency secretion of foreign proteins in yeast (Korea Patent No. 0626753, Korean Patent No. 0798894, Korean Patent No. 0975596). All discovered TFPs are functionally composed of a pre-secretion signal consisting of 20 amino acids at the amino terminus and a pro-secretion signal or secretion fusion partner connected thereto. It is. However, each TFP has a disadvantage that it is difficult to apply to the secretion production of a variety of foreign proteins, because the protein that can promote the secretion production is limited.

이에, 본 발명자들은 다양한 외래단백질을 보다 효과적으로 분비생산할 수 있는 방법을 개발하기 위하여, 예의 연구노력한 결과, 상기 TFP의 프리-분비시그널 및 프로-분비시그널을 랜덤으로 조합하여 구성된 인공 단백질 분비융합인자를 포함하는 라이브러리를 구성하고, 상기 라이브러리를 이용하면 난발현성의 외래 단백질의 분비생산에 적합한 TFP를 선별하여 외래단백질을 보다 용이하게 분비생산할 수 있음을 확인하고, 본 발명을 완성하였다.
Therefore, the inventors of the present invention, as a result of intensive research to develop a method that can effectively produce a variety of foreign proteins, as a result of the artificial protein secretion fusion factor consisting of a random combination of the pre-secretion signal and the pro-secretion signal of the TFP Comprising a library comprising, and using the library was confirmed that the TFP suitable for the secretion production of non-expressing foreign proteins can be more easily secreted production of foreign proteins, and completed the present invention.

본 발명의 목적은 효모 유래의 단백질 분비융합인자의 프리-분비시그널 및 프로-분비시그널을 포함하는 외래단백질의 분비생산용 인공 단백질 분비융합인자로서, 상기 프리-분비시그널과 프로-분비시그널은 서로 다른 단백질 분비융합인자로부터 유래되는 것인 인공 단백질 분비융합인자를 제공하는 것이다.An object of the present invention is an artificial protein secretion fusion factor for secretion production of foreign proteins, including a pre-secretion signal and a pro-secretion signal of yeast-derived protein secretion fusion factor, wherein the pre-secretion signal and pro-secretion signal are It is to provide an artificial protein secretion fusion factor that is derived from another protein secretion fusion factor.

본 발명의 다른 목적은 i) 상기 인공 단백질 분비융합인자 및 생산하고자 하는 외래 단백질 유전자를 포함하는 발현벡터를 제조하는 단계; ii) 상기 i) 단계의 발현벡터를 효모에 도입하여 형질전환체를 수득하는 단계; 및 iii) 상기 형질전환체를 배양하고, 이의 배양물 또는 배양상등액으로부터 외래단백질을 회수하는 단계를 포함하는, 외래 단백질을 생산하는 방법을 제공하는 것이다.Another object of the present invention is i) preparing an expression vector comprising the artificial protein secretion fusion factor and the foreign protein gene to be produced; ii) introducing the expression vector of step i) into yeast to obtain a transformant; And iii) culturing the transformant, and recovering the foreign protein from the culture or the culture supernatant thereof.

본 발명의 또 다른 목적은 (ⅰ) 상기 인공 단백질 분비융합인자를 포함하는 라이브러리에 목적하는 외래단백질의 유전자를 도입하여 각각의 발현벡터를 수득하는 단계; (ⅱ) 상기 각 발현벡터를 효모에 도입하여 각각의 형질전환체를 수득하는 단계; (ⅲ) 수득한 각 형질전환체를 배양하여 외래단백질을 분비생산하는 단계; (ⅳ) 분비생산된 외래단백질의 양에 따라 외래단백질의 분비생산 효율이 우수한 형질전환체를 선별하는 단계; 및 (ⅴ) 상기 선별된 형질전환체에 포함된 발현벡터를 분석하여 프리-분비시그널 및 프로-분비시그널의 조합을 결정하는 단계를 포함하는, 외래단백질의 분비생산에 적합한 인공 단백질 분비융합인자의 스크리닝 방법을 제공하는 것이다.
Still another object of the present invention is to obtain a respective expression vector by (i) introducing a gene of a foreign protein of interest into a library comprising the artificial protein secretion fusion factor; (Ii) introducing each of said expression vectors into yeast to obtain respective transformants; (Iii) culturing the obtained transformants to secrete and produce foreign proteins; (Iii) selecting a transformant having excellent secretion efficiency of the foreign protein according to the amount of the secreted foreign protein produced; And (iii) determining the combination of the pre-secretion signal and the pro-secretion signal by analyzing the expression vector included in the selected transformant, the artificial protein secretion fusion factor suitable for secretion production of foreign proteins. It is to provide a screening method.

상기의 목적을 달성하기 위한 하나의 실시양태로서, 본 발명은 효모 유래의 단백질 분비융합인자의 프리-분비시그널 및 프로-분비시그널을 포함하는 외래단백질의 분비생산용 인공 단백질 분비융합인자로서, 상기 프리-분비시그널과 프로-분비시그널은 서로 다른 단백질 분비융합인자로부터 유래되는 것인 인공 단백질 분비융합인자를 제공한다.
As one embodiment for achieving the above object, the present invention is an artificial protein secretion fusion factor for secretion production of foreign proteins comprising pre-secretion signal and pro-secretion signal of protein secretion fusion factor derived from yeast, The pre-secretion signal and the pro-secretion signal provide artificial protein secretion fusion factors that are derived from different protein secretion fusion factors.

본 발명의 용어 "단백질 분비융합인자(translational fusion partner, TFP)" 는 재조합 효모 발현 시스템에서 난발현성 단백질을 코딩하는 유전자와 융합되어 난발현성 단백질의 분비생산을 유도하는 유전자를 의미한다.　구체적으로, TFP-1, TFP-2, TFP-3, TFP-4 등을 열거할 수 있다(표 1).
The term "translational fusion partner (TFP)" of the present invention refers to a gene that is fused with a gene encoding a non-expressing protein in a recombinant yeast expression system to induce the secretion production of the non-expressing protein. Specifically, TFP-1, TFP-2, TFP-3, TFP-4 and the like can be enumerated (Table 1).

본 발명의 용어 "프리-분비시그널"이란, 세포에서 발현되는 단백질 중 합성과정 중에 소포체(endoplasmic reticulum)를 통과하는 단백질의 경우 대부분 단백질의 아미노 말단에 약 20여 개의 분비시그널을 가지고 있으며 이는 유전자 서열에 이미 존재하는 서열로서 단백질 합성 과정 중에 세포자체적으로 이러한 서열을 인식하고 합성된 단백질이 소포체로 유도되도록 하는 서열을 의미한다. 단백질 합성초기 합성된 아미노 말단이 소포체로 들어오게 되면 시그널 펩티다제(signal peptidase)에 의해 제거되는 20여 개의 단백질 서열이다. 효모에서 약 500여 개의 단백질이 소포체를 통과하는 단백질로 알려져 있다. 본 발명에서, 상기 프리-분비시그널은 특별히 이에 제한되지 않으나, TFP1-Pre(서열번호 1), TFP2-Pre(서열번호 2), TFP3-Pre(서열번호 3), TFP4-Pre(서열번호 4), TFP9-Pre(서열번호 5), TFP10-Pre(서열번호 6), TFP11-Pre(서열번호 7), TFP12-Pre(서열번호 8), TFP13-Pre(서열번호 9), TFP14-Pre(서열번호 10), TFP16-Pre(서열번호 11), TFP17-Pre(서열번호 12), TFP19-Pre(서열번호 13), TFP20-Pre(서열번호 14), TFP21-Pre(서열번호 15), TFP22-Pre(서열번호 16), TFP23-Pre(서열번호 17), TFP24-Pre(서열번호 18), TFP27-Pre(서열번호 19), TFP29-Pre(서열번호 20), TFP37-Pre(서열번호 21), TFP38-Pre(서열번호 22), TFP39-Pre(서열번호 23), TFP41-Pre(서열번호 24) 및 TFP42-Pre(서열번호 25)로 구성된 군으로부터 선택되는 단백질일 수 있으며, 또는 TFP1-Pre(서열번호 51), TFP2-Pre(서열번호 52), TFP3-Pre(서열번호 53), TFP4-Pre(서열번호 54), TFP9-Pre(서열번호 55), TFP10-Pre(서열번호 56), TFP11-Pre(서열번호 57), TFP12-Pre(서열번호 58), TFP13-Pre(서열번호 59), TFP14-Pre(서열번호 60), TFP16-Pre(서열번호 61), TFP17-Pre(서열번호 62), TFP19-Pre(서열번호 63), TFP20-Pre(서열번호 64), TFP21-Pre(서열번호 65), TFP22-Pre(서열번호 66), TFP23-Pre(서열번호 67), TFP24-Pre(서열번호 68), TFP27-Pre(서열번호 69), TFP29-Pre(서열번호 70), TFP37-Pre(서열번호 71), TFP38-Pre(서열번호 72), TFP39-Pre(서열번호 73), TFP41-Pre(서열번호 74) 및 TFP42-Pre(서열번호 75)로 구성된 군으로부터 선택되는 유전자로부터 발현되는 단백질일 수 있다.
The term “pre-secretory signal” of the present invention refers to a protein that passes through the endoplasmic reticulum during synthesis among proteins expressed in cells, and most have about 20 secretory signals at the amino terminus of a protein, which is a gene sequence. As a sequence already present in the protein refers to a sequence that recognizes this sequence in the cell itself during the protein synthesis process and the synthesized protein is induced into the endoplasmic reticulum. Protein Synthesis Initially, the synthesized amino termini is introduced into the endoplasmic reticulum, which is about 20 protein sequences removed by signal peptidase. About 500 proteins in yeast are known to pass through the endoplasmic reticulum. In the present invention, the pre-secretion signal is not particularly limited thereto, but TFP1-Pre (SEQ ID NO: 1), TFP2-Pre (SEQ ID NO: 2), TFP3-Pre (SEQ ID NO: 3), TFP4-Pre (SEQ ID NO: 4). ), TFP9-Pre (SEQ ID NO: 5), TFP10-Pre (SEQ ID NO: 6), TFP11-Pre (SEQ ID NO: 7), TFP12-Pre (SEQ ID NO: 8), TFP13-Pre (SEQ ID NO: 9), TFP14-Pre (SEQ ID NO: 10), TFP16-Pre (SEQ ID NO: 11), TFP17-Pre (SEQ ID NO: 12), TFP19-Pre (SEQ ID NO: 13), TFP20-Pre (SEQ ID NO: 14), TFP21-Pre (SEQ ID NO: 15) , TFP22-Pre (SEQ ID NO: 16), TFP23-Pre (SEQ ID NO: 17), TFP24-Pre (SEQ ID NO: 18), TFP27-Pre (SEQ ID NO: 19), TFP29-Pre (SEQ ID NO: 20), TFP37-Pre ( SEQ ID NO: 21), TFP38-Pre (SEQ ID NO: 22), TFP39-Pre (SEQ ID NO: 23), TFP41-Pre (SEQ ID NO: 24), and TFP42-Pre (SEQ ID NO: 25). Or TFP1-Pre (SEQ ID NO: 51), TFP2-Pre (SEQ ID NO: 52), TFP3-Pre (SEQ ID NO: 53), TFP4-Pre (SEQ ID NO: 54), TFP9-Pre (SEQ ID NO: 55), TFP10-Pr e (SEQ ID NO: 56), TFP11-Pre (SEQ ID NO: 57), TFP12-Pre (SEQ ID NO: 58), TFP13-Pre (SEQ ID NO: 59), TFP14-Pre (SEQ ID NO: 60), TFP16-Pre (SEQ ID NO: 61 ), TFP17-Pre (SEQ ID NO: 62), TFP19-Pre (SEQ ID NO: 63), TFP20-Pre (SEQ ID NO: 64), TFP21-Pre (SEQ ID NO: 65), TFP22-Pre (SEQ ID NO: 66), TFP23-Pre (SEQ ID NO: 67), TFP24-Pre (SEQ ID NO: 68), TFP27-Pre (SEQ ID NO: 69), TFP29-Pre (SEQ ID NO: 70), TFP37-Pre (SEQ ID NO: 71), TFP38-Pre (SEQ ID NO: 72) , TFP39-Pre (SEQ ID NO: 73), TFP41-Pre (SEQ ID NO: 74), and TFP42-Pre (SEQ ID NO: 75).

본 발명의 용어 "프로 분비시그널"이란, 상기한 소포체를 통과하는 단백질 중 소포체내에서 단백질의 폴딩 및 분비를 도와주는 역할을 하는 서열로서 단백질에 따라서 존재하는 경우가 있으며 모든 분비단백질에 존재하는 서열은 아니다. 분비과정을 거쳐 최종적으로 합성된 단백질에서는 존재하지 않는 서열이며 1차적으로 소포체에서 프리-분비시그널이 제거된 후 프로-분비시그널은 주로 골지체에서 최종적으로 제거된다. 선행 발명에서 개발된 TFP의 경우에 외래단백질을 효율적으로 분비하기 위해서 발굴된 효모 단백질 서열로서 프리-분비시그널을 제외한 나머지 부분을 프로-분비시그널 또는 분비 융합파트너(secretion fusion partner)로 정의하고 있다. 이때, 프로-분비시그널은 자연적으로 존재하는 서열일 경우도 있으며 원래는 프로-분비시그널이 아닌 구조단백질의 일부인 경우도 있으나 인위적으로 골지체에서 세포 내 프로세싱이 가능하도록 KEX2와 같은 프로테아제 인식서열을 도입하여 프로-분비시그널 역할을 하도록 가공된 경우도 있다. 본 발명에서, 상기 프로-분비시그널은 특별히 이에 제한되지 않으나, TFP1-Pro(서열번호 26), TFP2-Pro(서열번호 27), TFP3-Pro(서열번호 28), TFP4-Pro(서열번호 29), TFP9-Pro(서열번호 30), TFP10-Pro(서열번호 31), TFP11-Pro(서열번호 32), TFP12-Pro(서열번호 33), TFP13-Pro(서열번호 34), TFP14-Pro(서열번호 35), TFP16-Pro(서열번호 36), TFP17-Pro(서열번호 37), TFP19-Pro(서열번호 38), TFP20-Pro(서열번호 39), TFP21-Pro(서열번호 40), TFP22-Pro(서열번호 41), TFP23-Pro(서열번호 42), TFP24-Pro(서열번호 43), TFP27-Pro(서열번호 44), TFP29-Pro(서열번호 45), TFP37-Pro(서열번호 46), TFP38-Pro(서열번호 47), TFP39-Pro(서열번호 48), TFP41-Pro(서열번호 49) 및 TFP42-Pro(서열번호 50)로 구성된 군으로부터 선택되는 단백질, 또는 TFP1-Pro(서열번호 76), TFP2-Pro(서열번호 77), TFP3-Pro(서열번호 78), TFP4-Pro(서열번호 79), TFP9-Pro(서열번호 80), TFP10-Pro(서열번호 81), TFP11-Pro(서열번호 82), TFP12-Pro(서열번호 83), TFP13-Pro(서열번호 84), TFP14-Pro(서열번호 85), TFP16-Pro(서열번호 86), TFP17-Pro(서열번호 87), TFP19-Pro(서열번호 88), TFP20-Pro(서열번호 89), TFP21-Pro(서열번호 90), TFP22-Pro(서열번호 91), TFP23-Pro(서열번호 92), TFP24-Pro(서열번호 93), TFP27-Pro(서열번호 94), TFP29-Pro(서열번호 95), TFP37-Pro(서열번호 96), TFP38-Pro(서열번호 97), TFP39-Pro(서열번호 98), TFP41-Pro(서열번호 99) 및 TFP42-Pro(서열번호 100)로 구성된 군으로부터 선택되는 유전자로부터 발현되는 단백질일 수 있다.
The term "pro secretion signal" of the present invention is a sequence that serves to help the folding and secretion of the protein in the endoplasmic reticulum among the proteins passing through the above-mentioned endoplasmic reticulum may exist in accordance with the protein and present in all the secreted proteins Is not. It is a sequence that does not exist in the finally synthesized protein through the secretion process. After the pre-secretion signal is first removed from the endoplasmic reticulum, the pro-secretory signal is mainly removed from the Golgi apparatus. In the case of the TFP developed in the prior invention, the yeast protein sequence discovered to efficiently secrete foreign proteins is defined as a pro-secretion signal or secretion fusion partner, except for the pre-secretion signal. In this case, the pro-secretory signal may be a naturally occurring sequence, but may be part of a structural protein that is not a pro-secretory signal. However, a protease recognition sequence such as KEX2 may be introduced to enable intracellular processing in the Golgi apparatus. Sometimes it is engineered to act as a pro-secretion signal. In the present invention, the pro-secretion signal is not particularly limited thereto, but TFP1-Pro (SEQ ID NO: 26), TFP2-Pro (SEQ ID NO: 27), TFP3-Pro (SEQ ID NO: 28), TFP4-Pro (SEQ ID NO: 29) ), TFP9-Pro (SEQ ID NO: 30), TFP10-Pro (SEQ ID NO: 31), TFP11-Pro (SEQ ID NO: 32), TFP12-Pro (SEQ ID NO: 33), TFP13-Pro (SEQ ID NO: 34), TFP14-Pro (SEQ ID NO: 35), TFP16-Pro (SEQ ID NO: 36), TFP17-Pro (SEQ ID NO: 37), TFP19-Pro (SEQ ID NO: 38), TFP20-Pro (SEQ ID NO: 39), TFP21-Pro (SEQ ID NO: 40) , TFP22-Pro (SEQ ID NO: 41), TFP23-Pro (SEQ ID NO: 42), TFP24-Pro (SEQ ID NO: 43), TFP27-Pro (SEQ ID NO: 44), TFP29-Pro (SEQ ID NO: 45), TFP37-Pro ( SEQ ID NO: 46), TFP38-Pro (SEQ ID NO: 47), TFP39-Pro (SEQ ID NO: 48), TFP41-Pro (SEQ ID NO: 49), and TFP42-Pro (SEQ ID NO: 50), or a protein selected from TFP1 -Pro (SEQ ID NO: 76), TFP2-Pro (SEQ ID NO: 77), TFP3-Pro (SEQ ID NO: 78), TFP4-Pro (SEQ ID NO: 79), TFP9-Pro (SEQ ID NO: 80), TFP10-Pro (SEQ ID NO: Number 81), TFP11-Pro (SEQ ID NO: 82), TFP12-Pro (SEQ ID NO: 83), TFP13-Pro (SEQ ID NO: 84), TFP14-Pro (SEQ ID NO: 85), TFP16-Pro (SEQ ID NO: 86), TFP17 -Pro (SEQ ID NO: 87), TFP19-Pro (SEQ ID NO: 88), TFP20-Pro (SEQ ID NO: 89), TFP21-Pro (SEQ ID NO: 90), TFP22-Pro (SEQ ID NO: 91), TFP23-Pro (SEQ ID NO: 92), TFP24-Pro (SEQ ID NO: 93), TFP27-Pro (SEQ ID NO: 94), TFP29-Pro (SEQ ID NO: 95), TFP37-Pro (SEQ ID NO: 96), TFP38-Pro (SEQ ID NO: 97), TFP39- It may be a protein expressed from a gene selected from the group consisting of Pro (SEQ ID NO: 98), TFP41-Pro (SEQ ID NO: 99) and TFP42-Pro (SEQ ID NO: 100).

본 발명의 용어 "외래단백질"은, 숙주세포에서 정상적으로는 발현되지 않고, 숙주세포의 외부로부터 도입된 유전자에 의하여 발현되는 단백질을 의미한다. 본 발명에서는 인체 또는 다양한 생명체 유래의 단백질을 재조합 생산하고자 할 때 단백질 자체의 특성으로 인해서 대장균이나 효모 등의 숙주세포에서 재조합 발현 생산이 어려운 단백질을 의미한다. 바람직하게는 인간성장호르몬(hGH) 단백질 일 수 있으나 이에 한정되지 않으며, 숙주세포에서 재조합 생산이 가능하더라도 효모에서는 생산성이 낮아 경제성이 없는 다수의 단백질을 포함할 수 있다.　이러한 단백질들은, 이들로 한정되는 것은 아니지만, 혈청단백질(인자 VII, VIII 및 IX를 포함한 혈액인자), 면역글로불린, 사이토카인(인터류킨), α-, β- 및 γ-인터페론, 콜로니자극인자(GM-CSF), 혈소판 유도된 성장 인자(PDGF), 포스포리파제-활성화 단백질(PLAP), 인슐린, 종양 괴사 인자(TNF), 성장 인자(예, TGF-α 또는 TGF-β와 같은 조직 성장 인자 및 내피 성장 인자), 호르몬(소낭-자극 호르몬, 갑상선-자극 호르몬, 항이뇨 호르몬, 색소성 호르몬 및 부갑상선 호르몬, 황체분비호르몬 및 이의 유사체), 칼시토닌(calcitonin), 칼시토닌 유전자 관련 펩타이드(Calcitonin Gene Related Peptide,CGPR), 엔케팔린(enkephalin), 소마토메딘, 에리스로포이에틴, 시상하부 분비 인자, 프롤락틴, 만성 고나도트로핀, 조직 플라스미노겐 활성화제, 성장호르몬 분비 펩타이드(growth hormone releasing peptide; GHPR), 흉선 체액성 인자(thymic humoral factor; THF) 등이 포함된다. 또한, 이러한 단백질에는 효소를 포함할 것이며, 예로는 탄수화물-특이적 효소, 단백질분해 효소, 산화환원 효소, 트랜스퍼라제, 하이드롤라제, 라이아제, 이소머라제 및 리가제가 포함된다.구체적인 효소로는 이들로 한정하는 것은 아니지만 아스파라기나제, 아르기나제, 아르기닌 데아미나제, 아데노신 데아미나제, 과산화물 디스뮤타제, 엔도톡시나제, 카탈라제, 키모트립신, 리파제, 우리카제, 아데노신 디포스파타제, 티로시나제 및 빌리루빈 옥시다제를 들 수 있다. 탄수화물-특이적 효소의 예로는 글루코즈 옥시다제, 글루코다제, 갈락토시다제, 글루코세레브로시다제, 글루코우로니다제 등이 포함된다.
The term "foreign protein" of the present invention means a protein that is not normally expressed in a host cell, but is expressed by a gene introduced from the outside of the host cell. In the present invention, when it is intended to recombinantly produce a protein derived from the human body or various organisms, it means a protein that is difficult to produce recombinant expression in host cells such as E. coli or yeast due to the characteristics of the protein itself. Preferably, it may be a human growth hormone (hGH) protein, but is not limited thereto. Even though recombinant production is possible in a host cell, the yeast may include a plurality of proteins having low economical efficiency because of low productivity. These proteins include, but are not limited to, serum proteins (blood factors including factors VII, VIII and IX), immunoglobulins, cytokines (interleukin), α-, β- and γ-interferons, colony stimulating factors (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 TGF-α or TGF-β) and Endothelial growth factor), hormones (vesicle-stimulating hormone, thyroid-stimulating hormone, antidiuretic hormone, pigment hormone and parathyroid hormone, luteinizing hormone and 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 ptide (GHPR), thymic humoral factor (THF), and the like. Such proteins will also include enzymes, and examples include carbohydrate-specific enzymes, proteolytic enzymes, redox enzymes, transferases, hydrolases, lyases, isomerases, and ligases. Although not limited to these, asparaginase, arginase, arginine deaminase, adenosine deaminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine dephosphatase, tyrosinase and bilirubin Oxidase. Examples of carbohydrate-specific enzymes include glucose oxidase, glucosidase, galactosidase, glucocerebrosidase, glucoronidase, and the like.

본 발명의 구체적인 실시예에 따르면, 상기 외래단백질은 인간의 성장호르몬(hGH)이고, 상기 hGH를 분비생산하기에 적합한 인공 단백질 분비융합인자는 특별히 이에 제한되지 않으나, TFP20-Pre(서열번호 14 및 64), TFP3-Pre(서열번호 3 및 53) 또는 TFP14-Pre(서열번호 10 및 60)인 프리-분비시그널과 TFP19-Pro(서열번호 38 및 88) 또는 TFP24-Pro(서열번호 43 및 93)인 프로-분비시그널을 포함함이 바람직하다.
According to a specific embodiment of the present invention, the foreign protein is human growth hormone (hGH), artificial protein secretion fusion factor suitable for secreting the production of hGH is not particularly limited, TFP20-Pre (SEQ ID NO: 14 and 64), pre-secretion signals TFP3-Pre (SEQ ID NOs 3 and 53) or TFP14-Pre (SEQ ID NOs 10 and 60) and TFP19-Pro (SEQ ID NOs 38 and 88) or TFP24-Pro (SEQ ID NOs 43 and 93) It is preferred to include a pro-secretion signal).

본 발명의 용어 "인간 성장호르몬(human Growth Hormone, hGH)"은 뇌하수체전엽 호르몬 중 가장 많이 분비되는 단일쇄(single chain)의 단백질 호르몬으로서, 191개의 아미노산 잔기로 구성되는 분자량 약 22 kDa의 호르몬을 의미한다. 이는 뇌와 눈을 제외한 거의 모든 체내 조직 장기의 세포를 증식시켜 균형잡힌 성장을 촉진하며, 골성장으로 키가 커지고 골격근이 비대해지는 생리학적 작용을 가지고 있다.The term "human growth hormone (hGH)" of the present invention is a single-chain protein hormone secreted most of the anterior pituitary hormones, a hormone having a molecular weight of about 22 kDa composed of 191 amino acid residues. it means. It promotes balanced growth by proliferating cells of almost every organ in the body except brain and eyes, and has a physiological effect that increases bone growth and enlarges skeletal muscle.

본 발명의 구체적 실시예에서, 고농도의 hGH를 생산하기 위한 방법으로 단백질 분비융합인자를 사용하였으며, 상기 단백질 분비융합인자는 동 발명자들이 개발하여 특허 출원한 기술을 활용하여 얻을 수 있다(한국특허 제0626753 호, 한국특허 제0798894호 한국특허 제0975596호). 표 1에 상기 기술을 이용하여 발굴된 효모 유래의 다양한 분비 단백질 유전자 25종을 나타내었으며, 이들의 활용성을 더욱 증진시키기 위하여 25종의 프리-분비시그널과 25종의 프로-분비시그널을 다시 재조합한 단백질 분비융합인자 라이브러리를 제조하였다(실시예 1). 도 1에 상기 각 25종의 프리-분비시그널 및 프로-분비시그널이 조합된 인공 단백질 분비융합인자를이용한 라이브러리 구축 방법을 설명하는 모식도를 나타내었다.In a specific embodiment of the present invention, a protein secretion fusion factor was used as a method for producing a high concentration of hGH, and the protein secretion fusion factor can be obtained by utilizing the technology developed and patented by the inventors (Korean Patent No. 0626753, Korean Patent No. 0798894, Korean Patent No. 0975596). Table 1 shows 25 different secretory protein genes derived from yeast discovered using the above technique, and in order to further enhance their utility, 25 pre-secretory signals and 25 pro-secretory signals are recombined again. One protein secretion fusion factor library was prepared (Example 1). FIG. 1 is a schematic diagram illustrating a library construction method using artificial protein secretion fusion factors in which each of the 25 pre-secretion signals and the pro-secretion signal is combined.

또한, 제조된 인공 분비융합인자 라이브러리로부터 hGH를 고분비 생산하는 분비융합인자를 스크리닝 하는 방법을 개발하였다(실시예2). 도 2에 세포내 재조합 방법을 이용하여 제조된 인공 단백질분비융합인자 라이브러리로부터 hGH 고분비 생산 분비융합인자를 스크리닝하는 과정을 설명하는 모식도를 나타내었다.In addition, a method for screening a secretion fusion factor for high secretion production of hGH from the prepared artificial secretion factor library was developed (Example 2). Figure 2 shows a schematic diagram illustrating a process for screening hGH high secretion production secretion factor from an artificial protein secretion fusion factor library prepared using intracellular recombination method.

인간 성장호르몬 항체를 이용한 1차 스크리닝 결과, 웨스턴 브롯팅하여 강한 시그널을 보이는 28개의 균주를 선별하였으며(도 3), 선별된 28개의 균주를 SDS-PAGE를 통해서 2차 스크리닝하여 고분비 생산균주를 선택하였다(도 4 및 5). As a result of the primary screening using human growth hormone antibody, 28 strains showing strong signals were selected by Western blotting (FIG. 3), and the selected 28 strains were screened secondly through SDS-PAGE to produce high-secreting strains. Selected (FIGS. 4 and 5).

선택되어진 균주들을 염기서열분석 결과, 서로 다른 프리-분비시그널과 프로-분비시그널로 구성되어 있음을 확인하였고, 프리-분비시그널로는 ATG 27, CIS3 또는 EMP24이고, 프로-분비 시그널로는 MFa 또는 SCW4이 인간 성장호르몬의 고농도 분비에 효과적임을 확인하였다(표 2).
As a result of sequencing of the selected strains, it was confirmed that they consisted of different pre-secretion signals and pro-secretion signals. The pre-secretion signals were ATG 27, CIS3 or EMP24, and the pro-secretion signals were MFa or SCW4 was found to be effective in the high secretion of human growth hormone (Table 2).

본 발명의 다른 실시양태에 의하면, 본 발명은 (i) 상기 인공 단백질 분비융합인자 및 생산하고자 하는 외래 단백질 유전자를 포함하는 발현벡터를 제조하는 단계; (ii) 상기 (i) 단계의 발현벡터를 효모에 도입하여 형질전환체를 수득하는 단계; 및 (iii) 상기 형질전환체를 배양하고, 이의 배양물 또는 배양상등액으로부터 외래단백질을 회수하는 단계를 포함하는, 외래단백질을 생산하는 방법을 제공한다. According to another embodiment of the present invention, the present invention comprises the steps of (i) preparing an expression vector comprising the artificial protein secretion fusion factor and the foreign protein gene to be produced; (ii) introducing the expression vector of step (i) into yeast to obtain a transformant; And (iii) culturing the transformant, and recovering the foreign protein from the culture or the culture supernatant thereof.

이때, 상기 발현벡터는 특별히 이에 제한되지 않으나, 도 6의 개열지도로 표시되는 플라스미드 YGaC9-hGH를 사용함이 바람직하다. In this case, the expression vector is not particularly limited, but it is preferable to use the plasmid YGaC9-hGH represented by the cleavage map of FIG. 6.

바람직하게, 상기 형질전환체의 배양은 비-이온성 계면활성제, 보다 바람직하게는 폴리소르베이트(Polysorbate)(상표명 tween20) 또는 폴록사머(poloxamer)(상표명 폴록사머 188)를 포함하는 배지를 이용하여 수행할 수 있다. Preferably, the culturing of the transformant is carried out using a medium containing a non-ionic surfactant, more preferably polysorbate (trade name tween20) or poloxamer (trade name poloxamer 188). Can be done.

또한, 상기 외래단백질을 생산하는 방법은 회수한 외래단백질을 정제하는 단계를 추가로 포함할 수 있으며, 상기 외래단백질이 hGH인 경우 상기 외래단백질을 정제하는 위하여는 음이온 교환 크로마토 그래피, 소수성 상호작용 크로마토그래피, 음이온 교환 크로마토그래피 및 젤여과 크로마토그래피의 순서로 수행함이 바람직하다.
In addition, the method for producing the foreign protein may further comprise the step of purifying the recovered foreign protein, if the foreign protein is hGH in order to purify the foreign protein anion exchange chromatography, hydrophobic interaction chromatography Preference is given to performing in the order of chromatography, anion exchange chromatography and gel filtration chromatography.

본 발명의 용어 "발현벡터"는 통상 외래 DNA의 단편이 삽입된 캐리어로서 일반적으로 이중 가닥의 DNA의 단편을 의미한다. 여기서, 외래 DNA는 숙주 세포에서 천연적으로 발견되지 않는 DNA인 이종 DNA를 의미한다. 발현 벡터는 일단 숙주 세포내에 있으면 숙주 염색체 DNA와 무관하게 복제할 수 있으며 삽입된 외래 DNA가 발현될 수 있다. 당업계에 주지된 바와같이, 숙주 세포에서 형질감염 유전자의 발현 수준을 높이기 위해서는 해당 유전자가 선택된 발현 숙주 내에서 기능을 발휘하는 전사 및 해독 발현 조절 서열에 작동 가능하도록 연결되어야만 한다.The term "expression vector" of the present invention generally refers to a double stranded DNA fragment as a carrier into which a fragment of foreign DNA is inserted. 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.

본 발명의 용어 "형질전환"은 DNA를 숙주로 도입하여 DNA가 염색체외 인자로서 또는 염색체 통합완성에 의해 복제가능하게 되는 것을 의미한다. 본 발명에 따른 형질전환에 사용될 수 있는 숙주 세포는 원핵 또는 진핵 세포 모두를 포함할 수 있다. DNA의 도입효율이 높고, 도입된 DNA의 발현효율이 높은 숙주가 통상 사용된다.세균, 예를 들어 에스케리키아, 슈도모나스, 바실러스, 스트렙토마이세스, 진균, 효모와 같은 주지의 진핵 및 원핵 숙주들, 스포도프테라 프루기페르다(SF9)와 같은 곤충 세포, CHO, COS 1, COS 7, BSC 1, BSC 40, BMT 10 등의 동물 세포 등이다. 본 발명의 형질전환에 사용한 숙주세포는 캔디다(Candida), 디베리오마이세스(Debaryomyces), 한세눌라(Hansenula), 클루이베로마이세스(Kluyveromyces), 피키아(Pichia), 스키조사카로마이세스(Schizosaccharomyces), 야로이야(Yarrowia) 및 사카로마이세스(Saccharomyces)속 등의 효모류, 아스퍼질러스(Aspergillus), 페니실리엄(Penicillium), 라이조퍼스(Rhizopus) 및 트리코더마(Trichoderma)속 등의 균류 또는 에셔리키아(Escherichia) 및 바실러스(Bacillus) 속 등의 세균류를 사용할 수 있으나 반드시 이에 한정되는 것은 아니다.　　　　　　　　The term "transformation" of the present invention means that DNA is introduced into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration. Host cells that can be used for transformation according to the present invention can include both prokaryotic or eukaryotic cells. Hosts with high DNA transduction efficiency and high expression efficiency of introduced DNA are commonly used. Known eukaryotic and prokaryotic hosts such as bacteria, for example Escherichia, Pseudomonas, Bacillus, Streptomyces, fungi, yeast And insect cells such as Spodoptera pruperferda (SF9), and animal cells such as CHO, COS 1, COS 7, BSC 1, BSC 40, and BMT 10. The host cells used in the transformation of the present invention is Candida, Debaryomyces, Hansenula, Kluyveromyces, Pichia, Chizosaccharomyces Yeast, such as genus Yarrowia and Saccharomyces, fungi or eschers such as genus Aspergillus, Penicillium, Rhizopus and Trichoderma Bacteria, such as Escherichia and Bacillus, may be used, but are not necessarily limited thereto.

본 발명의 구체적 실시예에서는 프리-분비시그널 CIS3와 프로-분비시그널 MFa의 조합을 가지는 인공 단백질 분비융합인자와 hGH 유전자를 포함하는 발현벡터를 제조(도 6)하여, 이를 효모 사카로마이세스 세레비지에에 도입하여 형질전환체를 수득하였다.In a specific embodiment of the present invention to prepare an expression vector comprising an artificial protein secretion fusion factor and hGH gene having a combination of pre-secretion signal CIS3 and pro-secretion signal MFa (Fig. 6), this is the yeast Saccharomyces sere It was introduced into the busy cell to obtain a transformant.

또한, 본 발명의 hGH 생산방법은, 상기 형질전환된 효모를 유가식 발효 배양시켜 hGH 단백질을 생산 및 분비시키는 단계를 추가로 포함될 수 있다. In addition, the hGH production method of the present invention may further include the step of producing and secreting the hGH protein by fed-batch fermentation of the transformed yeast.

본 발명에서 "유가식 배양"은 배지를 간헐적으로 공급하는 배양방법으로서 배양액 중의 기질농도를 임의로 제어할 수 있으며, 기질은 적당한 속도로 첨가되며 유출이 없기 때문에 공급되는 기질의 양과 미생물에 의한 소비량 사이에 균형을 유지함으로써 기질을 자유롭게 제어할 수 있는 배양방법을 의미하며, 가장 보편화되어 있는 배양방법이다.
In the present invention, the "batch-fed culture" is a culture method for intermittently supplying the medium, which can arbitrarily control the substrate concentration in the culture solution, and because the substrate is added at an appropriate rate and there is no outflow, between the amount of substrate supplied and the consumption by the microorganisms. Means a culture method that can freely control the substrate by maintaining a balance in, is the most common culture method.

본 발명의 구체적 실시예에서, 초기배양 단계를 거친 200㎖의 최소배지를 배양하여 활성화 시킨 후, 5L 의 본 배양액에 접종하여 30시간 내지 48시간 동안 발효를 진행하였다(실시예 3). 배지에 분비된 hGH는 시간별로 시료를 취하여 600nm 파장에서 흡광도를 측정하였고(도 7의 A), 배양상등액을 SDS-PAGE 분석(도 7의 B)을 통해서 예상하는 크기의 인간 성장호르몬의 분비생산을 확인하였다. In a specific example of the present invention, after activating by culturing the minimum medium of 200ml after the initial culture step, and inoculated in 5L of the culture medium and proceeded with fermentation for 30 hours to 48 hours (Example 3). The hGH secreted in the medium was sampled over time to measure the absorbance at 600 nm wavelength (Fig. 7 A), the culture supernatant secretion production of human growth hormone of the size expected through SDS-PAGE analysis (B in Fig. 7) It was confirmed.

또한, 본 발명의 hGH 생산방법은 당업계에 잘 알려진 정제 방법이 추가적으로 포함될 수 있다. 예컨대, 면역친화성 크로마토그래피, 수용체친화성 크로마토그래피, 소수성 작용 크로마토그래피, 렉틴 친화성 크로마토그래피, 크기배제 크로마토그래피, 양이온 또는 음이온 교환 크로마토그래피, 고성능 액체 크로마토그래피(HPLC) 및 역상 HPLC 등을 포함하는 종래의 크로마토그래피 방법에 의해 숙주 세포가 자라는 배지에서 분리될 수 있다. 또한, 원하는 단백질이 특이적 태그, 라벨 또는 킬레이트 모이어티를 가지는 융합 단백질이어서 특이적 결합 파트너 또는 제제에 의해 인식하여 정제하는 방법이 있다. 정제된 단백질은 원하는 단백질 부분으로 절단되거나 그 자체로 남아있을 수 있다. 융합 단백질의 절단으로 절단 과정에서 부가적인 아미노산을 가지는 원하는 단백질 형태가 만들어질 수 있다.In addition, the hGH production method of the present invention may further include a purification method well known in the art. Examples include immunoaffinity chromatography, receptor affinity chromatography, hydrophobic action chromatography, lectin affinity chromatography, size exclusion chromatography, cation or anion exchange chromatography, high performance liquid chromatography (HPLC), reverse phase HPLC, and the like. The host cells can be separated from the medium in which the host cells are grown by conventional chromatography methods. In addition, there is a method in which the 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. Purified protein can be cleaved into the desired protein portion or left on its own. Cleavage of the fusion protein may result in the desired protein form having additional amino acids in the cleavage process.

상기에서 상술한 바와 같이 발현된 단백질의 특성에 따라, 종래 정제방법으로는 경제적인 대량생산을 수행하기 어려운 재조합 단백질은 정제공정의 최적화를 통하여 생산성을 향상시킬 수 있다.According to the characteristics of the protein expressed as described above, the recombinant protein difficult to perform economical mass production by the conventional purification method can improve productivity through optimization of the purification process.

또한, 본 발명의 hGH 생산방법은 고농도로 분비 생산된 hGH가 서로 결합하여 불용화되는 문제점을 해결하기 위하여 형질전환체를 유가식으로 배양할 때, 인체에 무해한 비-이온성 계면활성제인 Tween 20 또는 폴록사머(poloxamer)를 첨가하여 hGH를 활성화시키는 단계를 추가로 포함할 수 있다.In addition, the hGH production method of the present invention, Tween 20, a non-ionic surfactant that is harmless to the human body when culturing the transformant in a fed-batch to solve the problem that high-secreted hGH is insoluble by binding to each other. Alternatively, the method may further include activating hGH by adding a poloxamer.

본 발명에서 "비-이온성 계면활성제"는 물에서 이온으로 해리되지 않는 계면 활성제로서 분자중에 이온으로 해리되지 않는 수산기(-OH), 에테르결합(-O-), 아마이드결합(-CONH-), 에스테르결합(-COOR)등을 분자 중에 갖고있는 계면활성제을 의미한다. 비이온성 계면활성제의 친유기, 친수기의 밸런스차이에 따라 용해도, 습윤력, 침투력, 유화력, 가용화력등의 성질이 달라지며, 대표적으로 Tween20, poloxamer 가 있다.A "non-ionic surfactant" in the present invention is a surfactant that does not dissociate into ions in water and does not dissociate into ions in the molecule. It means a surfactant having a hydroxyl group (-OH), an ether bond (-O-), an amide bond (-CONH-), an ester bond (-COOR) and the like in the molecule. Solubility, wettability, penetration, emulsification, solubilization, etc. vary depending on the balance difference between the lipophilic and hydrophilic groups of nonionic surfactants. Tween20 and poloxamer are typical examples.

본 발명의 구체적 실시예에서, 상기 형질전환체를 유가식으로 배양발효시, 고농도로 분비 생산된 hGH가 서로 결합하여 불용화되는 문제점을 해결하기 위해, 배양시 비-이온성 계면활성제 Tween 20 및 폴록사머를 가하여, 이들의 효과 및 효모의 세포성장에 미치는 영향에 대해 알아보았다(실시예 4). 그 결과, 계면활성제 Tween 20 및 poloxamer은 세포의 생장에 치명적인 영향을 끼치지 않는다는 것을 확인하였고(도 8), SDS-PAGE를 통해 계면활성제의 농도가 높아질수록 불용성 단백질의 양이 줄어드는 것을 확인하였다(도 9).In a specific embodiment of the present invention, when fermenting the transformant in a fed-batch, in order to solve the problem that hGH produced in high concentration secretion is insoluble by binding to each other, non-ionic surfactant Tween 20 and Poloxamer was added to investigate the effects of these and effects on the cell growth of yeast (Example 4). As a result, it was confirmed that the surfactant Tween 20 and poloxamer did not have a fatal effect on the growth of the cells (Fig. 8), and the amount of insoluble protein was reduced as the concentration of the surfactant was increased through SDS-PAGE ( 9).

이렇게 발효 생산된 hGH를 정제하기 위해, 약 음이온 교환 크로마토 그래피(Anion Exchange Chromatography), 소수성 상호작용 크로마토그래피(Hydrophobic Interaction Chromatography), 2차 강 음이온 교환 크로마토그래피(Anion Exchange Chromatography), 젤 여과 크로마토그래피(Gel Filtration Chromatography)의 4단계 정제과정을 거쳐 hGH을 분리 정제하였다(실시예 5 및 도 10 내지 13).In order to purify the fermented hGH, weak anion exchange chromatography, hydrophobic interaction chromatography, secondary strong anion exchange chromatography, gel filtration chromatography ( HGH was separated and purified through a four step purification process of Gel Filtration Chromatography (Example 5 and FIGS. 10 to 13).

정제 단계별로 SDS-PAGE분석 및 웨스턴 블롯팅을 통해 재조합 hGH을 분석하였으며(실시예 6 및 도 14), 단백질 어세이 키트를 이용하여 브래드-포드 어세이 방법에 따라 단백질 정량하고, HPSEC를 통해 순도를 확인하였다. 그 결과, 순도 97%의 hGH 63㎎을 얻었다(표 3).Recombinant hGH was analyzed by SDS-PAGE analysis and Western blotting step by step (Example 6 and FIG. 14), protein quantification using a protein assay kit according to the Brad-Ford Assay method, and purity through HPSEC It was confirmed. As a result, 63 mg of hGH having a purity of 97% was obtained (Table 3).

끝으로, 정제된 hGH의 생물학적 활성을 확인하기 위해, Nb2 세포의 증식 효과를 측정함으로써 활성 분석하였다(실시예 7). 그 결과, 단백질의 Nb2 세포 증식활성은 대조구 인체 성장호르몬 활성과 유사하게 나타나는 것을 확인하였다(도 15).
Finally, in order to confirm the biological activity of the purified hGH, the activity was analyzed by measuring the proliferative effect of Nb2 cells (Example 7). As a result, it was confirmed that the Nb2 cell proliferation activity of the protein is similar to the control human growth hormone activity (Fig. 15).

본 발명의 또 다른 실시양태에 의하면, 본 발명은 외래단백질을 과발현시키기 위한, 인공 단백질 분비융합인자 라이브러리로부터 가장 효율이 뛰어난 프리-분비시그널 및 프로-분비시그널의 조합을 스크리닝하는 방법을 제공한다. According to another embodiment of the present invention, the present invention provides a method for screening the most efficient combination of pre-secretion signals and pro-secretory signals from an artificial protein secretion fusion factor library for overexpressing foreign proteins.

구체적으로, 상기 스크리닝하는 방법은 (ⅰ) 본 발명에 따른 인공 단백질 분비융합인자를 포함하는 라이브러리에 목적하는 외래단백질의 유전자를 도입하여 각각의 발현벡터를 수득하는 단계; (ⅱ) 상기 각 발현벡터를 효모에 도입하여 각각의 형질전환체를 수득하는 단계; (ⅲ) 수득한 각 형질전환체를 배양하여 외래단백질을 분비생산하는 단계; (ⅳ) 분비생산된 외래단백질의 양에 따라 외래단백질의 분비생산 효율이 우수한 형질전환체를 선별하는 단계; 및 (ⅴ) 상기 선별된 형질전환체에 포함된 발현벡터를 분석하여 프리-분비시그널 및 프로-분비시그널의 조합을 결정하는 단계를 포함한다.Specifically, the screening method includes the steps of: (i) introducing a gene of a foreign protein of interest into a library comprising an artificial protein secretion fusion factor according to the present invention to obtain respective expression vectors; (Ii) introducing each of said expression vectors into yeast to obtain respective transformants; (Iii) culturing the obtained transformants to secrete and produce foreign proteins; (Iii) selecting a transformant having excellent secretion efficiency of the foreign protein according to the amount of the secreted foreign protein produced; And (iii) determining the combination of the pre-secretion signal and the pro-secretion signal by analyzing the expression vector included in the selected transformant.

본 발명의 용어 "인공 단백질 분비융합인자 라이브러리"란 재조합 효모 발현 시스템에서 난발현 단백질의 분비생산을 유도하는 단백질 분비융합인자의 프리-분비시그널 및 프로-분비시그널을 랜덤으로 조합하여, 서로 다른 조합의 프리-분비시그널 및 프로-분비시그널로 구성된 인공 단백질 분비융합인자를 포함하는 라이브러리를 의미한다. 이들 유전자 라이브러리는 게놈성(염색체성) DNA 또는 cDNA 형태일 수 있다. 상기 라이브러리로부터 수득한 단백질 분비융합인자는, 대장균 등의 원핵세포와 효모 등의 진핵세포에서 재조합적 생산이 가능하지 않은 단백질뿐만 아니라, 대장균 등 다른 숙주에서 재조합 생산이 가능하더라도 효모와 같은 진핵세포에서는 생산성이 낮아 경제성이 없는 다수의 단백질을 재조합적으로 생산하는데 이용될 수 있다.
The term "artificial protein secretion fusion factor library" of the present invention refers to random combinations of pre-secretion signals and pro-secretory signals of protein secretion fusion factors that induce secretion production of the protein of expression in recombinant yeast expression systems. It means a library comprising an artificial protein secretion fusion factor consisting of the pre-secretion signal and the pro-secretion signal of. These gene libraries may be in the form of genomic (chromosomal) DNA or cDNA. The protein secretion fusion factor obtained from the library is not only a protein which is not capable of recombinant production in prokaryotic cells such as Escherichia coli and eukaryotic cells such as yeast, but also in eukaryotic cells such as yeast, although recombinant production is possible in other hosts such as E. coli. Low productivity can be used to recombinantly produce a large number of economical proteins.

본 발명의 프리-분비시그널과 프로-분비시그널의 신규한 조합을 가지는 인공 단백질 분비융합인자를 이용하면 hGH를 비롯한 종래의 재조합 기술에 의하여 효모에서 대량으로 생산하기 어려운 단백질을 대량으로 생산할 수 있으므로, 기존에 낮은 생산성으로 재조합 단백질 발현에 널리 사용되지 못한 효모 발현 시스템을 이용한 재조합 단백질의 생산에 널리 활용될 수 있을 것이다.
Artificial protein secretion fusion factor having a novel combination of pre-secretion signal and pro-secretion signal of the present invention can be produced in large quantities in a protein that is difficult to produce in yeast by conventional recombinant techniques, including hGH, It may be widely used for the production of recombinant protein using a yeast expression system that has not been widely used for expression of recombinant protein with low productivity.

도 1은 효모 유래의 분비단백질을 이용한 프리-분비시그널 및 프로-분비시그널이 조합된 인공 단백질 분비융합인자 라이브러리 구축 방법을 설명하는 모식도이다.
도 2는 세포내 재조합 방법을 이용하여 인공 단백질 분비융합인자 라이브러리로부터 인간 성장호르몬 고분비 생산 분비융합인자를 스크리닝하는 과정을 설명하는 모식도이다.
도 3은 형질전환체의 배양 상등액의 단백질이 결합된 나이트로셀룰로오스막을 대상으로 하여 인간 성장호르몬 항체를 이용한 도트 블롯팅을 수행한 결과를 나타내는 사진이다.
도 4는 선별된 28종의 형질전환체를 배양하여 배양상등액을 SDS-PAGE 분석한 결과를 보여주는 전기영동 사진이다.
도 5는 2차 선별된 균주를 고농도 배양하여 분비생산된 인간 성장호르몬의 분비량을 비교한 SDS-PAGE 결과를 보여주는 전기영동 사진이다.
도 6은 형질전환체 C9에 포함된 플라스미드 YGaC9-hGH의 개열지도이다.
도 7은 YGaC9-hGH가 도입된 형질전환체를 유가식으로 배양할 때의 세포의 성장(A) 및 시간별로 취한 배지를 SDS-PAGE로 분석한 결과(B)를 나타내는 그래프 및 전기영동 사진이다.
도 8은 비-이온계 계면활성제 tween20을 농도별로 첨가한 배지(A)와 폴록사머(poloxamer) 188을 첨가한 배지(B)에서의 재조합 효모 균주의 생장을 비교한 결과를 보여주는 그래프이다.
도 9는 비-이온계 계면활성제 tween20(A) 및 폴록사머 188(B)의 농도에 따른, hGH의 응축억제 효과를 분석한 결과를 나타내는 전기영동사진이다.
도 10은 정제시료를 1차 음이온 교환 크로마토그래피에 적용하여 수득한 활성분획의 전기영동 사진이다.
도 11은 1차 음이온 교환 크로마토그래피의 활성분획을 소수성 상호작용 크로마토그래피에 적용하여 수득한 활성분획의 전기영동 사진이다.
도 12는 소수성 상호작용 크로마토그래피의 활성분획을 2차 음이온 교환 크로마토그래피에 적용하여 수득한 활성분획의 전기영동 사진이다.
도 13은 2차 음이온 교환 크로마토그래피의 활성분획을 젤 여과 크로마토그래피에 적용하여 수득한 활성분획의 전기영동 사진이다.
도 14는 정제 단계별 단백질 분획에 대한 SDS-PAGE 및 웨스턴블롯팅 결과를 보여주는 사진이다.
도 15는 hGH의 생물학적 활성확인을 위하여 농도에 따른 Nb2 세포 증식 결과를 보여주는 그래프이다.1 is a schematic diagram illustrating a method for constructing an artificial protein secretion fusion factor library in which a pre-secretion signal and a pro-secretion signal using a secreted protein derived from yeast are combined.
Figure 2 is a schematic diagram illustrating a process for screening human growth hormone high secretion production secretion factor from the artificial protein secretion factor library using intracellular recombination method.
3 is a photograph showing the results of dot blotting using a human growth hormone antibody targeting the nitrocellulose membrane to which the protein of the culture supernatant of the transformant is bound.
Figure 4 is an electrophoresis picture showing the results of the SDS-PAGE analysis of the culture supernatant by culturing the selected 28 transformants.
Figure 5 is an electrophoresis picture showing the results of SDS-PAGE comparing the secretion amount of human growth hormone secreted by the high concentration of the secondary screened strain.
6 is a cleavage map of plasmid YGaC9-hGH contained in transformant C9.
Figure 7 is a graph and electrophoresis picture showing the results of the growth of cells (A) and the time taken by the SDS-PAGE analysis of the culture (B) when culturing the YGaC9-hGH-introduced transformant fed in a fed-batch manner (B) .
FIG. 8 is a graph showing the results of comparison of the growth of recombinant yeast strains in medium (A) to which non-ionic surfactant tween20 is added at different concentrations and to medium (B) to which poloxamer 188 is added.
Figure 9 is an electrophoresis picture showing the results of analyzing the condensation inhibitory effect of hGH according to the concentration of the non-ionic surfactant tween20 (A) and poloxamer 188 (B).
10 is an electrophoretic photograph of an active fraction obtained by applying a purified sample to primary anion exchange chromatography.
11 is an electrophoretic photograph of an active fraction obtained by applying an active fraction of primary anion exchange chromatography to hydrophobic interaction chromatography.
12 is an electrophoretic photograph of an active fraction obtained by applying an active fraction of hydrophobic interaction chromatography to secondary anion exchange chromatography.
FIG. 13 is an electrophoretic photograph of an active fraction obtained by applying an active fraction of secondary anion exchange chromatography to gel filtration chromatography.
Figure 14 is a photograph showing the SDS-PAGE and Western blotting results for the protein fractions in each step of purification.
15 is a graph showing the results of Nb2 cell proliferation according to the concentration to confirm the biological activity of hGH.

이하, 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시 예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

실시예Example 1:  One: 단백질분비융합인자Protein Secretion Fusion Factor 조합을 통한 인공  Artificial through combination 단백질분비융합인자Protein Secretion Fusion Factor 라이브러리의 제조 Preparation of the Library

효모에서 외래단백질을 고효율 분비생산하는 방법으로 본 발명자들은 효모 유전체로부터 다양한 분비 단백질 유전자를 확보하고 외래단백질 맞춤형 분비 융합인자(translational fusion partner, TFP) 기술을 개발하였다(한국특허 제0626753호, 한국특허 제0798894호 및 한국특허 제0975596호). 상기 기술에서 발굴된 효모유래의 다양한 분비 단백질 유전자 25종은 표 1에 표시된 바와 같다.
As a method of producing high-efficiency secretion of foreign proteins in yeast, the present inventors have secured various secretory protein genes from the yeast genome and developed a technology for the production of a translational fusion partner (TFP) tailored to foreign proteins (Korean Patent No. 0626753, Korean Patent). No. 0798894 and Korean Patent No. 0975596). 25 kinds of yeast-derived various secreted protein genes discovered in the above technique are shown in Table 1.

효모 유래의 다양한 TFPVariety of TFP from Yeast TFPTFP ORF(유전자)명   ORF name 기능function TFP1TFP1 YAR066WYAR066W 기능미확인 GPI proteinUnidentified GPI protein TFP2TFP2 YFR026C(ULI1)YFR026C (ULI1) ER내 unfolded protein response 관련 단백질 Unfolded protein response related protein in ER TFP3TFP3 YJL158C(CIS3)YJL158C (CIS3) 세포벽 PIR 단백질Cell wall PIR protein TFP4TFP4 Hansenula polymorpa HpPrB Hansenula polymorpa HpPrB Vacuole 단백분해효소 BVacuole Protease B TFP9TFP9 YGR106C(VOA1)YGR106C (VOA1) vacuolar ATPase 결합인자 vacuolar ATPase binding factor TFP10TFP10 YOR247W(SRL1)YOR247W (SRL1) 세포벽 mannoproteinCell wall mannoprotein TFP11TFP11 YIL123W(SIM1)YIL123W (SIM1) SUN family (Sim1p, Uth1p, Nca3p, Sun4p) 단백질SUN family (Sim1p, Uth1p, Nca3p, Sun4p) proteins TFP12TFP12 YOR085W(OST1) YOR085W (OST1) 소포체 oligosaccharyltransferase complex 감마 subunitEndoplasmic reticulum oligosaccharyltransferase complex gamma subunit TFP13TFP13 YNL190WYNL190W 기능미확인 세포벽 단백질Unidentified cell wall protein TFP14TFP14 YGL200C(EMP24) YGL200C (EMP24) 단백질수송 관련 ER 막단백질Protein Transport Related ER Membrane Proteins TFP16TFP16 YJL159W(HSP150) YJL159W (HSP150) heat shock proteinheat shock protein TFP17TFP17 YBR078W(ECM33) YBR078W (ECM33) 기능미확인 GPI 단백질Unidentified GPI Protein TFP19TFP19 YPL187W(MFalpha1)YPL187W (MFalpha1) 교배인자 alphaCrossing factor alpha TFP20TFP20 YJL178C(ATG1) YJL178C (ATG1) Autophagy 관련 막단백질Autophagy Related Membrane Proteins TFP21TFP21 YKR042W(UTH1) YKR042W (UTH1) 미토콘드리아 외막단백질Mitochondrial outer membrane protein TFP22TFP22 YDR077W(SED1) YDR077W (SED1) stress 유도 GPI 단백질stress-induced GPI protein TFP23TFP23 YGR282C(BGL2) YGR282C (BGL2) Endo-beta-1,3-glucanaseEndo-beta-1,3-glucanase TFP24TFP24 YGR279C(SCW4) YGR279C (SCW4) 세포벽단백질Cell wall proteins TFP27TFP27 YLR110C(CCW12) YLR110C (CCW12) 세포벽 합성관여 세포벽단백질Cell wall protein TFP29TFP29 YOR383C(FIT3)YOR383C (FIT3) 세포벽 mannoproteinCell wall mannoprotein TFP37TFP37 YNL160W(YGP1) YNL160W (YGP1) 세포벽 관련 분비 단백질Cell Wall Related Secretory Proteins TFP38TFP38 YLR390W-A(CCW14) YLR390W-A (CCW14) 세포벽 이온결합 당단백질Cell Wall Ionic Glycoproteins TFP39TFP39 YLR037C(PAU23) YLR037C (PAU23) 세포벽 mannoproteinCell wall mannoprotein TFP41TFP41 YHR139C(SPS100) YHR139C (SPS100) 스포아 형성 단백질Spore forming proteins TFP42TFP42 YOL155C(HPF1)YOL155C (HPF1) Haze-protective mannoproteinHaze-protective mannoprotein

발굴된 모든 TFP는 기능적으로 아미노말단에 17 내지 24개의 아미노산으로 구성된 프리-분비시그널(pre-secretion signal)(서열번호 1 내지 25)과 그의 카르복시 말단에 연결된 44 내지 202개의 아미노산으로 구성된 프로-분비시그널(pro-secretion signal)(서열번호 26 내지 50) 또는 분비 융합파트너(secretion fusion partner)로 구성되어 있다. 이렇게 확보된 단백질 분비융합인자는 효모 자체에 자연적으로 존재하는 서열이며 다양한 외래단백질의 분비를 촉진하는 역할을 수행하였다.
All discovered TFPs are functionally pre-secretion signals consisting of 17 to 24 amino acids at the amino terminus (SEQ ID NOS: 1 to 25) and pro-secretions consisting of 44 to 202 amino acids linked to their carboxy termini. Pro-secretion signal (SEQ ID NO: 26 to 50) or secretion fusion partner (secretion fusion partner). The protein secretion fusion factor thus obtained is a sequence naturally present in the yeast itself, and played a role in promoting the secretion of various foreign proteins.

본 발명에서는 이들의 활용성을 더욱 증진시키기 위하여 25종의 프리-분비시그널의 유전자(TFP1-Pre 내지 TFP42-Pre, 서열번호 51 내지 75)와 25종의 프로-분비시그널의 유전자(TFP1-Pro 내지 TFP42-Pro, 서열번호 76 내지 100)를 다시 재조합한 인공 단백질 분비융합인자 라이브러리를 제조하였다.
In the present invention, 25 kinds of pre-secretion signal genes (TFP1-Pre to TFP42-Pre, SEQ ID NOs: 51 to 75) and 25 kinds of pro-secretory signal genes (TFP1-Pro) in order to further enhance their utility. To TFP42-Pro, SEQ ID NO: 76 to 100) to prepare a recombinant protein secretion factor library.

이를 위해 25종의 TFP 유전자를 포함하는 효모벡터 YGa-GAL-TFP를 주형으로 하고, 센스 프라이머로는 공통적으로 GAL100(서열번호 101)을 사용하고, 각 TFP에 대해 프리-분비시그널을 중합효소 연쇄반응을 통해 증폭할 수 있는 안티센스 프라이머, T1-H170(서열번호 102), T2-H171(서열번호 103), T3-H172(서열번호 104), T4-H173(서열번호 105), T9-H174(서열번호 106), T10-H175(서열번호 107), T11-H176(서열번호 108), T12-H177(서열번호 109), T13-H178(서열번호 110), T14-H179(서열번호 111), T16-H180(서열번호 112), T17-H181(서열번호 113), T19-H182(서열번호 114), T20-H183(서열번호 115), T21-H184(서열번호 116), T22-H185(서열번호 117), T23-H186(서열번호 118), T24-H187(서열번호 119), T27-H188(서열번호 120), T29-H189(서열번호 121), T37-H190(서열번호 122), T38-H191(서열번호 123), T39-H192(서열번호 124), T41-H193(서열번호 125) 및 T42-H194(서열번호 126)을 사용한 PCR을 수행하여 각 TFP 유래의 프리-분비시그널을 증폭하였다(94℃에서 5분 동안 1회; 94℃ 30초간, 55℃ 30초간, 72℃ 3분간, 72℃ 1분간 반응을 25회; 72℃에서 7분간 1회).
To this end, the yeast vector YGa-GAL-TFP containing 25 TFP genes is used as a template, and GAL100 (SEQ ID NO: 101) is commonly used as a sense primer, and a pre-secretion signal is linked to a polymerase chain for each TFP. Antisense primers that can be amplified by reaction, T1-H170 (SEQ ID NO: 102), T2-H171 (SEQ ID NO: 103), T3-H172 (SEQ ID NO: 104), T4-H173 (SEQ ID NO: 105), T9-H174 ( SEQ ID NO: 106), T10-H175 (SEQ ID NO: 107), T11-H176 (SEQ ID NO: 108), T12-H177 (SEQ ID NO: 109), T13-H178 (SEQ ID NO: 110), T14-H179 (SEQ ID NO: 111), T16-H180 (SEQ ID NO: 112), T17-H181 (SEQ ID NO: 113), T19-H182 (SEQ ID NO: 114), T20-H183 (SEQ ID NO: 115), T21-H184 (SEQ ID NO: 116), T22-H185 (SEQ ID NO: 117), T23-H186 (SEQ ID NO: 118), T24-H187 (SEQ ID NO: 119), T27-H188 (SEQ ID NO: 120), T29-H189 (SEQ ID NO: 121), T37-H190 (SEQ ID NO: 122), T38 -H191 (SEQ ID NO: 123), T39-H192 (SEQ ID NO: 124), T41-H193 (SEQ ID NO: 125), and T42-H194 (SEQ ID NO: PCR was carried out using 126) to amplify the pre-secretion signal derived from each TFP (once for 5 minutes at 94 ° C; for 94 ° C for 30 seconds, for 55 ° C for 30 seconds, for 72 ° C for 3 minutes, and for 72 ° C for 1 minute). 25 times; once at 72 ° C. for 7 minutes).

또한, 프로-분비시그널 부분을 따로 증폭하기 위해서 25종의 TFP 유전자를 포함하는 효모벡터 YGa-GAL-TFP를 주형으로 하고, 각 TFP에 대한 센스프라이머 T1-H225(서열번호 127), T2-H226(서열번호 128), T3-H227(서열번호 129), T4-H228(서열번호 130), T9-H229(서열번호 131), T10-H230(서열번호 132), T11-H231(서열번호 133), T12-H232(서열번호 134), T13-H233(서열번호 135), T14-H234(서열번호 136), T16-H235(서열번호 137), T17-H236(서열번호 138), T19-H237(서열번호 139), T20-H238(서열번호 140), T21-H239(서열번호 141), T22-H240(서열번호 142), T23-H241(서열번호 143), T24-H242(서열번호 144), T27-H243(서열번호 145), T29-H244(서열번호 146), T37-H245(서열번호 147), T38-H246(서열번호 148), T39-H247(서열번호 149), T41-H248(서열번호 150) 및 T42-H249(서열번호 151)와 공통 안티센스 프라이머로 GT90R(서열번호 152)를 사용한 PCR을 수행하여 각 TFP에 대한 프로-분비시그널을 증폭하였다(94℃에서 5분 동안 1회; 94℃ 30초간, 55℃ 30초간, 72℃ 3분간, 72℃ 1분간 반응을 25회; 72℃에서 7분간 1회).
In addition, a yeast vector YGa-GAL-TFP containing 25 TFP genes was used as a template to separately amplify the pro-secretory signal portion, and the sense primers T1-H225 (SEQ ID NO: 127) and T2-H226 for each TFP were used. (SEQ ID NO: 128), T3-H227 (SEQ ID NO: 129), T4-H228 (SEQ ID NO: 130), T9-H229 (SEQ ID NO: 131), T10-H230 (SEQ ID NO: 132), T11-H231 (SEQ ID NO: 133) , T12-H232 (SEQ ID NO: 134), T13-H233 (SEQ ID NO: 135), T14-H234 (SEQ ID NO: 136), T16-H235 (SEQ ID NO: 137), T17-H236 (SEQ ID NO: 138), T19-H237 ( SEQ ID NO: 139), T20-H238 (SEQ ID NO: 140), T21-H239 (SEQ ID NO: 141), T22-H240 (SEQ ID NO: 142), T23-H241 (SEQ ID NO: 143), T24-H242 (SEQ ID NO: 144), T27-H243 (SEQ ID NO: 145), T29-H244 (SEQ ID NO: 146), T37-H245 (SEQ ID NO: 147), T38-H246 (SEQ ID NO: 148), T39-H247 (SEQ ID NO: 149), T41-H248 (SEQ ID NO: 150) and T42-H249 (SEQ ID NO: 151) and PCR using GT90R (SEQ ID NO: 152) as a common antisense primer to each TFP. Amplified pro-secretion signal (once for 5 minutes at 94 ° C .; 25 times for 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 3 minutes, 72 ° C. for 1 minute; once at 72 ° C. for 7 minutes) .

증폭된 25종의 프리-분비시그널 유전자와 25종의 프로-분비시그널 유전자를 아가로스젤에서 전기영동하고 각각의 유전자를 젤로부터 회수한 다음 동량씩 혼합액을 만들었다. 이 혼합액에 폴리뉴클레오티드 키나아제를 이용하여 말단을 인산화시킨 후, 랜덤으로 라이게이션시켰다. 25 amplified pre-secretion signal genes and 25 pro-secretory signal genes were electrophoresed in agarose gel, and each gene was recovered from the gel, and then the same amount was prepared. The mixture was phosphorylated using polynucleotide kinase and then ligated randomly.

그런 다음, 이를 다시 프라이머 GAL100(서열번호 101)과 GT70r(서열번호 153)을 이용하여 증폭하였고, 증폭된 유전자를 제한효소 BamHI/SalI 또는 EcoRI/SalI 으로 처리하여 BamHI/SalI 또는 EcoRI/SalI으로 처리된 YGa 벡터에 삽입하였으며, 이를 대장균에 형질전환하여 약 1x104개의 형질전환체를 얻어, 인공 단백질 분비융합인자 라이브러리를 제조하였다(도 1). 도 1은 효모 유래의 분비단백질을 이용한 프리-분비시그널 및 프로-분비시그널이 조합된 인공 단백질 분비융합인자 라이브러리 구축 방법을 설명하는 모식도이다.
Then, it was amplified again using primers GAL100 (SEQ ID NO: 101) and GT70r (SEQ ID NO: 153), and the amplified gene was treated with restriction enzymes BamHI / SalI or EcoRI / SalI and treated with BamHI / SalI or EcoRI / SalI. It was inserted into the YGa vector, which was transformed into Escherichia coli to obtain about 1 × 10 4 transformants to prepare an artificial protein secretion fusion factor library (FIG. 1). 1 is a schematic diagram illustrating a method for constructing an artificial protein secretion fusion factor library in which a pre-secretion signal and a pro-secretion signal using a secreted protein derived from yeast are combined.

실시예Example 2: 인공 단백질  2: artificial protein 분비융합인자Secretion fusion factor 라이브러리로부터 외래단백질 분비  Foreign protein secretion from the library 융합인자의Fusion factor 선별 Selection

실시예 1에서 제조된 인공 단백질 분비융합인자 라이브러리로부터 외래단백질을 고분비 생산하는 분비융합인자를 스크리닝하는 방법을 개발하기 위하여, 인간 성장호르몬(human growth hormone, hGH)을 타겟 단백질로 사용하였다.Human growth hormone (hGH) was used as a target protein in order to develop a method for screening a secretion fusion factor for producing high secretion of foreign proteins from the artificial protein secretion fusion factor library prepared in Example 1.

hGH 유전자를 인공 단백질 분비융합인자 라이브러리에 도입하기 위해서 SH32(서열번호 154)와 SH33(서열번호 155)을 이용하여 hGH 유전자를 1차 PCR 하고, LNK39(서열번호 156)와 GT90R(서열번호 152)을 이용하여 2차 PCR 한 후 SwaI으로 처리된 인공 단백질 분비융합인자 라이브러리 벡터와 함께 효모 사카로마이세스 세레비지에 균주에 형질전환하고, 상기 형질전환체를 우라실이 없는 배지에서 배양하였다(도 2). 도 2는 세포내 재조합 방법을 이용하여 인공 단백질 분비융합인자 라이브러리로부터 인간 성장호르몬 고분비 생산 분비융합인자를 스크리닝하는 과정을 설명하는 모식도이다.
In order to introduce the hGH gene into the artificial protein secretion fusion factor library, first PCR was performed on the hGH gene using SH32 (SEQ ID NO: 154) and SH33 (SEQ ID NO: 155), and LNK39 (SEQ ID NO: 156) and GT90R (SEQ ID NO: 152). After the second PCR using a recombinant protein secretion fusion factor library vector treated with SwaI in yeast Saccharomyces cerevisiae strain was transformed, and the transformants were cultured in a medium without uracil (Fig. 2). ). Figure 2 is a schematic diagram illustrating a process for screening human growth hormone high secretion production secretion factor from the artificial protein secretion factor library using intracellular recombination method.

상기 우라실이 없는 배지에서 성장한 형질전환체를 200여개를 무작위적으로 선별하여 YPDG(효모추출물 1%, 펩톤 2%, 글루코스 1%, 갈락토스 1%) 배지가 첨가된 96 웰 플레이트에 접종하여 48시간 동안 진탕배양한 후, 배양상등액을 96웰 플레이트 필터 시스템을 이용하여 나이트로셀룰로오스 막에 통과시켜 배지 중 단백질을 나이트로셀룰로오스막에 결합시켰다. 이어 상기 막을 대상으로 하여 인간 성장호르몬 항체를 이용한 도트 블롯팅(dot-blotting)을 수행하였다.More than 200 transformants grown in the uracil-free medium were randomly selected and inoculated in 96 well plates to which YPDG (1% yeast extract, 2% peptone, 1% glucose, 1% galactose) medium was added for 48 hours. After shaking for a while, the culture supernatant was passed through a nitrocellulose membrane using a 96 well plate filter system to bind the protein in the medium to the nitrocellulose membrane. Then, the membrane was subjected to dot blotting using human growth hormone antibody.

구체적으로, 배지중의 단백질이 결합된 나이트로셀룰로오스막을 반응 버퍼(3% Bovine serum albumin을 함유하는 TBS 용액)에 넣어 상온에서 1시간 동안 블로킹하였다. 세척액(TBS 용액)으로 5분씩 3회 세척하고, 1차 마우스 유래의 단클론 항체 (Anti-human GH antibody, R&D systems)를 0.5% BSA가 함유된 TBS 버퍼에 5000배 희석하여 상온에서 1시간 동안 반응시키고, 세척액(TBS 용액)으로 5분씩 3회 세척하여 비특이적인 항체결합을 제거하였다. 이후 2차 마우스 IgG 항체(sigma)를 0.5% BSA+TBS 버퍼에 1000배 희석한 용액으로 상온에서 1시간 동안 반응이 끝난 후, 세척액으로 3회 세척한 후 멤브레인에 BCIP+NBP (sigma)기질을 첨가하여, 발색이 되는 것을 통해 hGH를 확인하였다(도 3). 도 3은 효모 형질전환체의 배양 상등액의 단백질이 결합된 나이트로셀룰로오스막을 대상으로 하여 인간 성장호르몬 항체를 이용한 도트 블롯팅을 수행한 결과를 나타내는 사진이다. 도 3에서 보듯이, 강한 시그널을 보이는 28개의 균주를 1차적으로 선별할 수 있었다.
Specifically, the nitrocellulose membrane to which the protein in the medium was bound was placed in the reaction buffer (TBS solution containing 3% Bovine serum albumin) and blocked for 1 hour at room temperature. After washing three times with a washing solution (TBS solution) three times, the primary mouse-derived monoclonal antibody (Anti-human GH antibody, R & D systems) was diluted 5000 times in TBS buffer containing 0.5% BSA and reacted at room temperature for 1 hour. Then, washed three times for 5 minutes with a washing solution (TBS solution) to remove nonspecific antibody binding. After completion of the reaction for 1 hour at room temperature with a solution diluted 1000-fold in 0.5% BSA + TBS buffer, the secondary mouse IgG antibody (sigma) was washed three times with a wash solution, and then the BCIP + NBP (sigma) substrate was added to the membrane. Addition, hGH was confirmed through the development of color (Fig. 3). 3 is a photograph showing the results of dot blotting using a human growth hormone antibody targeting the nitrocellulose membrane to which the protein of the culture supernatant of the yeast transformant is bound. As shown in Figure 3, 28 strains showing a strong signal could be selected primarily.

상기 선별된 28개의 균주를 다시 테스트 튜브에 배양하고, 배양 상등액을 농축 후 SDS-PAGE하여 hGH를 고분비 생산하는 균주를 선별하였다(도 4). 도 4는 선별된 28종의 형질전환체를 배양하여 배양상등액을 SDS-PAGE 분석한 결과를 보여주는 전기영동 사진이다. 도 4에서 보듯이, hGH를 고분비 생산하는 4종의 균주(C3, C9, C16 및 C28)를 2차 적으로 선별할 수 있었고, 상기 선별된 4종의 균주에 함유된 플라스미드를 분리하고 그의 염기서열을 분석하였다(표 2).
The selected 28 strains were cultured again in a test tube, and the culture supernatant was concentrated, and then SDS-PAGE was performed to select strains that produce high secretion of hGH (FIG. 4). Figure 4 is an electrophoresis picture showing the results of the SDS-PAGE analysis of the culture supernatant by culturing the selected 28 transformants. As shown in FIG. 4, four strains (C3, C9, C16 and C28) that produce high secretion of hGH were able to be secondarily selected, and the plasmids contained in the selected four strains were isolated and The base sequence was analyzed (Table 2).

2차 선별된 형질전환체의 플라스미드에 포함된 프리-분비시그널과 프로-분비시그널 분석결과Analysis of pre-secretion signal and pro-secretion signal included in plasmid of secondary screened transformant 형질전환체Transformant 프리분비시그널Pre-secretion signal 프로분비시그널Pro secretion signal C3C3 TFP20-Pre(ATG27)TFP20-Pre (ATG27) TFP19-Pro(MFa)TFP19-Pro (MFa) C9C9 TFP3-Pre(CIS3)TFP3-Pre (CIS3) TFP19-Pro(MFa)TFP19-Pro (MFa) C16C16 TFP14-Pre(EMP24)TFP14-Pre (EMP24) TFP19-Pro(MFa)TFP19-Pro (MFa) C28C28 TFP14-Pre(EMP24)TFP14-Pre (EMP24) TFP24-Pro(SCW4)TFP24-Pro (SCW4)

상기 표 2에서 보듯이. 상기 선별된 4종의 균주는 서로 다른 프리-분비시그널과 프로-분비시그널로 구성되어 있음을 확인하였고, 이 중 3개가 동일한 프로-분비시그널인 TFP19-Pro(서열번호 88)(MFa, mating factor alpha)를 함유하여 인간 성장호르몬의 고농도 분비를 위해서는 TFP19(MFa)의 프로-분비시그널이 사용하는 것이 필수적임을 확인하였다.
As shown in Table 2 above. The selected four strains were confirmed to be composed of different pre-secretion signals and pro-secretion signals, three of which are the same pro-secretion signal TFP19-Pro (SEQ ID NO: 88) (MFa, mating factor It was confirmed that the pro-secretory signal of TFP19 (MFa) is essential for the high concentration of human growth hormone containing alpha).

각 균주로부터 분리된 단일균체 3개를 선별하여 다시 각각 고농도 배양하여 분석한 결과, 도 5에서 보는 바와 같이 C9이 가장 높은 단백질 분비율을 보여 hGH 생산을 위한 최종 균주로 선택하였다.
As a result of selecting three single cells isolated from each strain and culturing them again in high concentration, as shown in FIG. 5, C9 showed the highest protein secretion ratio and was selected as the final strain for hGH production.

실시예Example 3: 인간 성장호르몬의 발효 생산  3: Fermentation Production of Human Growth Hormone

실시예 2에서 최종 선택된 형질전환체 C9를 이용하여 인간 성장호르몬을 대량 생산하고자, 상기 형질전환체 C9로부터 TFP3-Pre(서열번호 53)와 TFP19-Pro(서열번호 88)로 구성된 인공 TFP 유전자와 hGH 유전자를 포함하는 플라스미드 YGaC9-hGH를 추출하였다(도 6). 도 6은 형질전환체 C9에 포함된 플라스미드 YGaC9-hGH의 개열지도이다.
In order to mass produce human growth hormone using the transformant C9 finally selected in Example 2, an artificial TFP gene consisting of TFP3-Pre (SEQ ID NO: 53) and TFP19-Pro (SEQ ID NO: 88) from the transformant C9; The plasmid YGaC9-hGH containing the hGH gene was extracted (FIG. 6). 6 is a cleavage map of plasmid YGaC9-hGH contained in transformant C9.

상기 추출한 플라스미드 YGaC9-hGH를 효모 사카로마이세스 세레비지에 균주에 도입하여 형질전환체를 제조하고, 이를 유가식으로 배양하였다.The extracted plasmid YGaC9-hGH was introduced into a strain in yeast Saccharomyces cerevisiae to prepare a transformant, and cultured by fed-batch.

구체적으로, 본 배양에 들어가기 전에 상기 형질전환체를 50㎖ YNB(0.67% 아미노산이 결여된 효모기질, 0.5% 카사미노에시드, 2% 글루코스)배지에서 초도 배양한 다음, 다시 200㎖의 YEPD 액체 배지(Yeast extract 10g/ℓ, Peptone 20g/ℓ, Dextrose 20g/ℓ)에서 배양하고, 배양한 균주를 1.8L 발효배지(2% 포도당, 4% 효모추출물, 1% 펩톤)가 포함된 5L 규모의 발효기에 접종하였다. 포도당이 완전히 소모되면 공급배지(30% 포도당, 30% 갈락토스, 15% 효모추출물)를 균체의 성장에 따라 시간당 2g/L에서 20g/L로 점차로 추가하면서 30 내지 48시간동안 유가식으로 배양하였다.
Specifically, the transformants were first cultured in 50 ml YNB (yeast substrate lacking 0.67% amino acid, 0.5% cassamino acid, 2% glucose) medium before entering the main culture, and then again 200 ml of YEPD liquid medium. (Yeast extract 10g / l, Peptone 20g / l, Dextrose 20g / l), and the cultured strain was a 5L fermenter containing 1.8L fermentation medium (2% glucose, 4% yeast extract, 1% peptone) Was inoculated. When glucose was completely consumed, the feed medium (30% glucose, 30% galactose, 15% yeast extract) was incubated for 30-48 hours in a fed-batch for 30 to 48 hours while gradually adding from 2 g / L to 20 g / L per hour as the growth of the cells.

배양된 형질전환체에서 생산되어 배지로 분비된 hGH의 생산량을 측정하기 위하여, 상기 배지시료를 시간별로 취하여 600nm 파장에서 흡광도를 측정하고, 배양상등액 10㎕을 SDS-PAGE 분석하여 hGH의 분비생산 정도를 확인하였다(도 7). 도 7은 YGaC9-hGH가 도입된 형질전환체를 유가식으로 배양할 때의 세포의 성장(A) 및 시간별로 취한 배지를 SDS-PAGE로 분석한 결과(B)를 나타내는 그래프 및 전기영동 사진이다. 그 결과, 상기 형질전환체가 hGH를 약 600 mg/ℓ의 수준의 고농도로 분비생산할 수 있음을 확인하였다. In order to measure the production amount of hGH produced in the cultured transformant and secreted into the medium, the media samples were taken at different times and measured for absorbance at 600 nm wavelength. It was confirmed (Fig. 7). Figure 7 is a graph and electrophoresis picture showing the results of the growth of cells (A) and the time taken by the SDS-PAGE analysis of the culture (B) when culturing the YGaC9-hGH-introduced transformant fed in a fed-batch manner (B) . As a result, it was confirmed that the transformant can secrete and produce high concentrations of hGH at a level of about 600 mg / l.

다만, 고농도로 분비생산된 hGH는 발효 및 농축과정에서 서로 결합하여 응축되어 불활성화된 상태로 존재하기 때문에, 이를 그대로 사용할 수 없다는 문제점이 새로이 제기되었다.
However, the hGH produced at high concentration is condensed by being combined with each other in the fermentation and concentration process, so that it cannot be used as it is.

실시예Example 4:  4: 비이온성Nonionic 계면활성제의 효과 Effect of Surfactants

상기 실시예 3의 결과에서 보듯이, hGH를 고농도로 분비생산할 수는 있었으나, 이들이 불활성화된 상태로 존재하기 때문에, 계면활성제를 처리하여 이를 활성화시키고자 하였다.As shown in the result of Example 3, although hGH can be secreted and produced at a high concentration, since they existed in an inactivated state, the surfactant was treated to activate it.

비이온성 계면활성제인 tween20(sigma) 또는 폴록사머 188(sigma)을 농도별(0%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8% 또는 1%(w/v))로 배지에 첨가하여, 형질전환체의 성장 및 이로부터 분비생산되는 hGH의 응축에 미치는 영향을 확인하였다. Nonionic surfactant tween20 (sigma) or poloxamer 188 (sigma) is added to the medium by concentration (0%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8% or 1% (w / v)) By doing so, the effect on the growth of the transformant and the condensation of hGH secreted and produced therefrom was confirmed.

구체적으로, 본 배양에 들어가기 전에 상기 형질전환체를 3㎖ YPD(2% 펩톤, 1% 효모기질, 2% 글루코스)배지에서 초도 배양하고, 배양물을 상기 tween20 또는 폴록사머 188이 농도별(0%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8% 또는 1%(w/v))로 첨가된 50㎖의 YPDG 배지(2% 펩톤, 1% 효모기질, 1% 갈락토스, 1% 글루코스)에 접종하여 30 내지 60시간 동안 배양하였다. 시간별로 시료를 취하여 세포의 성장 정도를 600nm 파장에서 흡광도를 측정하여 계면활성제가 세포의 생장에 치명적인 영향을 끼치지 않는 것을 확인하였다(도 8). 도 8은 비-이온계 계면활성제인 tween20을 농도별로 첨가한 배지(A)와 폴록사머(poloxamer)를 첨가한 배지(B)에서의 형질전환체의 생장을 비교한 결과를 나타내는 그래프이다. 도 8에서 보듯이, 비-이온계 계면활성제인 tween20 또는 폴록사머 188은 형질전환체의 생장에 별다른 영향을 미치지 않음을 알 수 있었다.
Specifically, the transformants were initially cultured in 3 ml YPD (2% peptone, 1% yeast substrate, 2% glucose) medium before entering the main culture, and the culture was determined by concentration of tween20 or poloxamer 188 (0). 50 ml of YPDG medium (2% peptone, 1% yeast substrate, 1% galactose, 1% glucose) added in%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8% or 1% (w / v) ) Was incubated for 30 to 60 hours. Samples were taken by time to measure the absorbance at 600 nm wavelength of the growth rate of the cells to confirm that the surfactant does not have a fatal effect on the growth of the cells (Fig. 8). FIG. 8 is a graph showing the results of comparison of the growth of transformants in the medium (A) to which tween20, a non-ionic surfactant, is added for each concentration, and the medium (B) to which poloxamer is added. As shown in Figure 8, it can be seen that the non-ionic surfactant tween20 or poloxamer 188 does not significantly affect the growth of the transformant.

한편, 분비생산된 hGH의 응축이 상기 비-이온계 계면활성제에 의하여 억제되는지의 여부를 확인하기 위하여, 상기 배양된 각 배양물을 원심분리하여 각각의 배양상등액을 수득하고, 이를 15,000rpm, 4℃ 조건에서 30분동안 원심분리하여 얻은 침전물을 각각 SDS-PAGE 분석하였다(도 9). 도 9는 비-이온계 계면활성제 tween20(A) 및 폴록사머 188(B)의 농도에 따른, hGH의 응축억제 효과를 분석한 결과를 나타내는 전기영동사진이다. 도 9에서 보듯이, 계면활성제의 농도가 증가할 수록 불용성 단백질(insoluble protein)인 응축된 hGH의 함량이 감소됨을 알 수 있었다. 다만, 상기 계면활성제를 사용하여도 hGH의 응축을 완전히 억제할 수는 없었고, 0.4%(w/v) 이상의 농도로 사용할 경우에는 별다른 효과를 나타내지 못하였으므로, 이후에서는 상기 계면활성제를 0.5%(w/v)의 농도로 사용하였다.
On the other hand, in order to confirm whether the condensation of secreted and produced hGH is inhibited by the non-ionic surfactant, by centrifuging each culture of the culture to obtain the respective culture supernatant, which is 15,000rpm, 4 The precipitates obtained by centrifugation at 30 ° C. for 30 minutes were analyzed by SDS-PAGE, respectively (FIG. 9). Figure 9 is an electrophoresis picture showing the results of analyzing the condensation inhibitory effect of hGH according to the concentration of the non-ionic surfactant tween20 (A) and poloxamer 188 (B). As shown in Figure 9, it was found that as the concentration of the surfactant increases, the content of condensed hGH, an insoluble protein, decreases. However, even when the surfactant was used, it was not possible to completely inhibit the condensation of hGH, and when used at a concentration of 0.4% (w / v) or higher, it did not show any effect. / v).

실시예Example 5: 재조합  5: recombination 인간성장호르몬의Human growth hormone 분리정제 Separation tablet

분비생산된 hGH를 정제하기 위하여, 상기 배양물로부터 배양상등액을 수득하고, 이를 다양한 크로마토그래피에 순차적으로 적용하였다.
In order to purify the secreted hGH, a culture supernatant was obtained from the culture, which was sequentially applied to various chromatography.

실시예Example 5-1: 정제시료의 수득 5-1: Obtaining a Purified Sample

선별된 효모 Y2805(YGa-C9-hGH) 균주를 상기 실시예 3에서 설명한 바와 같은 유가식 발효배양시 모든 배지에 0.5%의 폴록사머 188을 첨가한 상태에서 발효하고 이로부터 얻어진 배양물을 원심분리하여(3,500rpm, 20min, 4℃) 배양상등액을 수득하고, 상기 배양상등액을 0.1 ㎛ 크기의 여과막으로 여과하여 균체를 완전히 제거한 다음, 한외여과(분자량 30kDa cut-off)를 수행하여 배양상등액을 농축하고, 이에 20mM 트리스 버퍼(pH8.0)를 가하여 정제시료를 수득하였다.
Selected yeast Y2805 (YGa-C9-hGH) strains were fermented in a fed-batch fermentation culture as described in Example 3 with 0.5% of poloxamer 188 added to all media and the culture obtained therefrom was centrifuged. (3,500rpm, 20min, 4 ℃) to obtain a culture supernatant, the culture supernatant was filtered through a 0.1 ㎛ filter membrane to completely remove the cells, and ultrafiltration (molecular weight 30kDa cut-off) to concentrate the culture supernatant Then, 20mM Tris buffer (pH8.0) was added thereto to obtain a purified sample.

실시예Example 5-2: 1차 음이온 교환  5-2: primary anion exchange 크로마토 그래피Chromatography

상기 실시예 5-1에서 수득한 정제시료를 미리 20mM 트리스 버퍼(pH8.0)로 평형시킨 DEAE Sepharose(GE healthcare) 컬럼 (5X10cm)에 10㎖/min의 속도로 흡착시켰다. 이어, 컬럼에 다시 20mM 트리스 버퍼(pH8.0)를 통과시켜서 컬럼내에 유리된 상태로 남아있는 단백질을 모두 제거하였다. The purified sample obtained in Example 5-1 was adsorbed on a DEAE Sepharose (GE healthcare) column (5 × 10 cm) previously equilibrated with 20 mM Tris buffer (pH 8.0) at a rate of 10 mL / min. Then, 20 mM Tris buffer (pH 8.0) was passed through the column again to remove all remaining protein in the column.

20mM 트리스 버퍼(pH8.0)과 0.5M Nacl이 함유된 20mM 트리스 버퍼(pH8.0)를이용하여 0부터 0.5M까지의 NaCl 직선농도 구배로 흡착된 단백질을 1ℓ용출시키고, 이를 SDS-PAGE로 확인하였다(도 10). 도 10은 정제시료를 1차 음이온 교환 크로마토그래피에 적용하여 수득한 활성분획의 전기영동 사진이다. 도 10에서 보듯이, hGH가 0.2M NaCl 농도에서 대부분 용출됨을 확인할 수 있었다.
Using a 20 mM Tris buffer (pH8.0) containing 20 mM Tris buffer (pH8.0) and 0.5 M Nacl, 1 L of the adsorbed protein was dissolved in a linear gradient of NaCl from 0 to 0.5 M. It was confirmed (FIG. 10). 10 is an electrophoretic photograph of an active fraction obtained by applying a purified sample to primary anion exchange chromatography. As shown in Figure 10, it was confirmed that hGH is mostly eluted at a concentration of 0.2M NaCl.

실시예Example 5-3: 소수성 상호작용 크로마토그래피 5-3: Hydrophobic Interaction Chromatography

상기 실시예 5-2에서 수득한 활성분획을 모아서 1M 암모늄설페이트 농도가 되도록 조절하여 용액 및 단백질의 소수성을 증가시켰다. 상기 소수성을 증가시킨 활성분획을, 1M 암모늄설페이트가 첨가된 20mM 트리스 버퍼로 평형화된 Phenyl sepharose fast flow(GE healthcare) 컬럼(2.2X11cm)에 가하여 목적 단백질을 흡착시켰다. 그런 다음, 20mM 트리스 버퍼와 1M 암모늄설페이트 첨가된 20mM 트리스 버퍼로 1M부터 0M 까지의 암모늄설페이트를 직선농도 구배로 흡착된 단백질의 소수성을 약화시키는 조건으로 400㎖를 용출시켰다 (도 11). 도 11은 1차 음이온 교환 크로마토그래피의 활성분획을 소수성 상호작용 크로마토그래피에 적용하여 수득한 활성분획의 전기영동 사진이다. 도 11에서 보듯이, hGH가 0M의 암모늄설페이트 농도에서 대부분 용출됨을 확인할 수 있었다.
The active fractions obtained in Example 5-2 were collected and adjusted to 1M ammonium sulfate concentration to increase the hydrophobicity of the solution and protein. The active fraction with increased hydrophobicity was added to a Phenyl sepharose fast flow (GE healthcare) column (2.2 × 11 cm) equilibrated with 20 mM Tris buffer with 1 M ammonium sulfate to adsorb the target protein. Then, 400 ml was eluted with 20 mM Tris buffer and 20 mM Tris buffer to which 1 M ammonium sulfate was added, under conditions that weakened the hydrophobicity of the adsorbed protein in a linear gradient of 1 M to 0 M (FIG. 11). 11 is an electrophoretic photograph of an active fraction obtained by applying an active fraction of primary anion exchange chromatography to hydrophobic interaction chromatography. As shown in FIG. 11, it was confirmed that hGH was mostly eluted at a concentration of 0M ammonium sulfate.

실시예Example 5-4: 2차 음이온 교환 크로마토그래피 5-4: Secondary Anion Exchange Chromatography

상기 실시예 5-3에서 수득한 활성분획을 모아서 암모늄설페이트를 투석하여 탈염하고, 20mM 트리스 버퍼(pH8.0)로 평형화된 Q 컬럼(GE healthcare : 1.1X12cm)에 흡착시킨 후, 컬럼에 다시 트리스 버퍼를 통과시켜 컬럼 내에 유리된 상태로 남아있는 단백질을 모두 제거하였다. 20mM 트리스 버퍼(pH8.0)와 0.5M NaCl이 함유된 20mM 트리스 버퍼(pH8.0)를 이용하여 0부터 0.5M까지의 NaCl 직선농도 구배로 흡착된 단백질을 120㎖ 용출시켰다(도 12). 도 12는 소수성 상호작용 크로마토그래피의 활성분획을 2차 음이온 교환 크로마토그래피에 적용하여 수득한 활성분획의 전기영동 사진이다. 도 12에서 보듯이, 0.2 내지 0.3M Nacl 농도에서 대부분 용출됨을 확인할 수 있었다.
The active fractions obtained in Example 5-3 were collected and desalted by dialysis of ammonium sulfate, adsorbed onto a Q column (GE healthcare: 1.1 × 12 cm) equilibrated with 20 mM Tris buffer (pH8.0), and then trised again on the column. The buffer was passed through to remove any protein that remained free in the column. Using 20 mM Tris buffer (pH8.0) and 20 mM Tris buffer (pH8.0) containing 0.5M NaCl, 120ml of the adsorbed protein was eluted with a NaCl linear concentration gradient from 0 to 0.5M (Fig. 12). 12 is an electrophoretic photograph of an active fraction obtained by applying an active fraction of hydrophobic interaction chromatography to secondary anion exchange chromatography. As shown in Figure 12, it was confirmed that the most eluted at a concentration of 0.2 to 0.3M Nacl.

실시예Example 5-5: 젤 여과 크로마토그래피 5-5: Gel Filtration Chromatography

상기 실시예 5-4에서 수득한 활성분획을 모아서 한외여과 방법(Amicon, 분자량 cut-off 10KDa)을 이용하여 농축한 다음, 이를 Superdex 75(1.6X61.5cm) 컬럼에 적용하여 젤 여과 크로마토그래피를 수행하였다. The active fractions obtained in Example 5-4 were collected and concentrated using an ultrafiltration method (Amicon, molecular weight cut-off 10KDa), which was then applied to a Superdex 75 (1.6X61.5cm) column to perform gel filtration chromatography. Was performed.

단백질의 크기별로 분리가 되는 방법으로 NaCl이 함유된 20mM 트리스버퍼를 1㎖/min의 속도로 통과시키면서 대략 60분 정도부터 나타난 피크를 SDS-PAGE로 분석하였다(도 13). 도 13은 2차 음이온 교환 크로마토그래피의 활성분획을 젤 여과 크로마토그래피에 적용하여 수득한 활성분획의 전기영동 사진이다. 도 13에서 보듯이 22 KDa의 hGH이 정제되었음을 확인하였다.
The peaks appearing from about 60 minutes were analyzed by SDS-PAGE while passing the 20 mM Trisbuffer containing NaCl at a rate of 1 ml / min in a method of separating proteins by size (FIG. 13). FIG. 13 is an electrophoretic photograph of an active fraction obtained by applying an active fraction of secondary anion exchange chromatography to gel filtration chromatography. As shown in FIG. 13, it was confirmed that 22 GH hGH was purified.

실시예Example 6: 정제된 재조합  6: purified recombination hGHhGH 의 단백질 분석Protein analysis

상기 실시예 5에서 정제한 재조합 hGH의 특성을 다양한 방법으로 분석하였다.
The recombinant hGH purified in Example 5 was analyzed by various methods.

실시예Example 6-1:  6-1: SDSSDS -- PAGEPAGE 및  And 웨스턴Western 블럿Blot

hGH의 정제과정에서 수득한 각 시료를 5㎍ 또는 10㎍씩 취하여 비환원 조건의 PAGE 또는 12% SDS-PAGE를 수행하여 22KDa의 hGH을 확인하고, 전기영동이 끝난 후 전개된 각각의 젤에 포함된 단백질을 전기블럿터(electroblotter, Bio-rad)를 이용하여 300mA에서 40분간 나이트로셀룰로오스막(Whatman)으로 이동시켜 웨스턴 블럿을 수행하였다. 5 ㎍ or 10 ㎍ of each sample obtained during the purification of hGH was performed by PAGE or 12% SDS-PAGE under non-reducing conditions to confirm 22 KDa of hGH, and included in each gel developed after electrophoresis. The prepared protein was transferred to a nitrocellulose membrane (Whatman) at 300 mA for 40 minutes using an electroblotter (Bio-rad) to perform Western blotting.

구체적으로, 상기 단백질이 전이된 NC막을 반응 버퍼(3% 소혈청 알부민을 함유하는 TBS 용액)에 넣어 상온에서 1시간 동안 블로킹하였다. 세척액(TBS 용액)으로 5분씩 3회 세척하고, 1차 마우스 유래의 단클론 항체 (Anti-human GH antibody, R&D systems)를 0.5% BSA가 함유된 TBS 버퍼에 5000배 희석하여 상온에서 1시간 동안 반응시키고, 세척액(TBS 용액)으로 5분씩 3회 세척하여 비특이적인 항체결합을 제거하였다. 이후 2차 마우스 IgG 항체(Sigma)를 0.5% BSA를 포함한 TBS 버퍼에 1000배 희석한 용액으로 상온에서 1시간 동안 반응이 끝난 후, 세척액으로 3회 세척한 후, 멤브레인에 BCIP+NBP (sigma)기질을 첨가하여, 발색이 되는 것을 통해 hGH을 확인하였다 (도 14). 도 14은 hGH의 정제단계별 시료를 대상으로 전기영동 및 웨스턴 블럿을 수행한 결과를 나타내는 사진으로서, 레인 1은 hGH 표준품, 레인 2는 발효농축액, 레인 3은 DEAE 분획, 레인 4는 소수성 상호작용 크로마토그래피(HIC) 분획, 레인 5는 음이온교환 크로마토그라피(AEC) 분획, 레인 6과 7은 gel filtration 분획을 나타낸다.
Specifically, the NC membrane to which the protein was transferred was blocked in a reaction buffer (TBS solution containing 3% bovine serum albumin) and blocked for 1 hour at room temperature. After washing three times with a washing solution (TBS solution) three times, the primary mouse-derived monoclonal antibody (Anti-human GH antibody, R & D systems) was diluted 5000 times in TBS buffer containing 0.5% BSA and reacted at room temperature for 1 hour. Then, washed three times for 5 minutes with a washing solution (TBS solution) to remove nonspecific antibody binding. The secondary mouse IgG antibody (Sigma) was then diluted 1000-fold in a TBS buffer containing 0.5% BSA at room temperature for 1 hour, and then washed three times with a wash solution, followed by BCIP + NBP (sigma). Substrate was added to confirm hGH by developing color (FIG. 14). 14 is a photograph showing the results of electrophoresis and Western blotting on the samples of the hGH purification step, lane 1 is a hGH standard, lane 2 is a fermentation concentrate, lane 3 is a DEAE fraction, lane 4 is a hydrophobic interaction chromatograph. The HIC fraction, lane 5 is the anion exchange chromatography (AEC) fraction, and lanes 6 and 7 are the gel filtration fractions.

실시예Example 6-2: 단백질 정량 6-2: Protein Quantitation

각 단계별로 얻은 단백질 용출 분획은 모두 단백질 어세이 키트(bio-rad) 을 이용한 브래드포드 어세이 방법에 따라서 BSA로 표준곡선을 작성하고, 595nm에서 측정된 흡광도를 통해 단백질 농도를 계산하였다(표 3).
The protein elution fractions obtained in each step were prepared by BSA according to the Bradford assay method using a protein assay kit (bio-rad), and the protein concentration was calculated by absorbance measured at 595 nm (Table 3). ).

각 정제단계별 단백질 농도Protein concentration for each purification step 정제단계Purification stage VolVol ..
(㎖)(Ml) ConcConc ..
(( mgmg .㎖).Ml) TotalTotal proteinprotein
(( mgmg )) StepStep YieldYield
(%)(%) OverallOverall YieldYield
(%)(%) PurityPurity
(%)(%) Ultra filtrationUltra filtration 130130 18.318.3 24002400 100100 100100 -- Ion exchange chromatography
(DE㎍AE)Ion exchange chromatography
(DEµAE) 100100 4.544.54 454454 1919 1919 67.767.7 Hydrophobic interaction chromatography
(Phenyl)Hydrophobic interaction chromatography
(Phenyl) 6565 2.922.92 190190 41.941.9 88 85.185.1 Ion exchange chromatography
(Q)Ion exchange chromatography
(Q) 2020 4.984.98 99.699.6 52.452.4 4.24.2 88.288.2 Gel filtration chromatography
(Superdex 75)Gel filtration chromatography
(Superdex 75) 1414 4.484.48 62.862.8 63.0563.05 2.62.6 9797

실시예Example 6-3:  6-3: HighHigh PressurePressure SizeSize ExclusionExclusion ChromatographyChromatography ( ( HPSECHPSEC ) 를 통한 순도확인Purity through)

TSKgel G2000SWXL(7.8X300mm, Tosoh) 컬럼에 5㎍(20㎕) 시료를 주입하고 25mM 암모늄 중탄산염(pH7.0)을 1㎖/min의 속도로 HPLC한 결과 95% 이상의 순도를 확인하였다. 상기 표 3에서 보듯이, 최종적으로 순도가 97%의 재조합 hGH를 약 63㎎을 회수할 수 있었다.
A 5 μg (20 μl) sample was injected into a TSKgel G2000SWXL (7.8 × 300 mm, Tosoh) column, and 25 mM ammonium bicarbonate (pH 7.0) was HPLC at a rate of 1 mL / min. As shown in Table 3, finally, about 63 mg of recombinant hGH with 97% purity could be recovered.

실시예Example 6-4: N-말단 서열분석 6-4: N-terminal sequencing

정제된 단백질을 이용하여 아미노 말단 서열분석을 한국기초과학지원연구원에 의뢰하여 분석하였다. 각 정제 단백질을 SDS-PAGE하고, 이를 PVDF막으로 옮기기 위해 일렉트로-트랜스퍼 키트를 이용하였으며, 10% 메탄올이 첨가된 CAPS(N-cyclohexyl-3- aminopropanesulfonic acid) 버퍼에서 300mA로 40분 동안 전기를 흘려 단백질을 옮겼다. 단백질의 트랜스퍼가 완료된 PVDF 막은 쿠마시블루로 염색하고 50% 메탄올로 탈색한 후 자연 건조시켰다. Amino terminal sequencing using the purified protein was analyzed by the Korea Institute of Basic Science. SDS-PAGE each purified protein and use an electro-transfer kit to transfer it to the PVDF membrane, flowing electricity at 300 mA for 40 minutes in N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer with 10% methanol. The protein was transferred. Protein transfer complete PVDF membranes were stained with Coomassie blue, bleached with 50% methanol and dried naturally.

이렇게 준비된 시료는 에드만 분해법(Edman degradation)을 이용한 서열분석장치를 통해 N말단 5개 아미노산을 분석하였다. The sample thus prepared was analyzed for five amino acids in the N terminal through a sequencing device using Edman degradation.

그 결과, 사람의 뇌하수체에서 정제한 인간 성장호르몬과 동일한 서열(FPTIP)이 확인되어 효모에서 재조합 생산된 인간 성장호르몬이 단백질 분비융합인자와 융합상태에서 분비과정 중에 정확히 프로세싱되어 완벽한 단백질이 제조되었음을 확인하였다.
As a result, the same sequence (FPTIP) as the human growth hormone purified from the human pituitary gland was identified, and the human growth hormone recombinantly produced in yeast was correctly processed during secretion in the state of fusion with the protein secretion fusion factor to confirm that a perfect protein was produced. It was.

실시예Example 7: 정제된  7: refined hGHhGH 의 세포기반 활성 측정Cell-based activity

정제된 인간 성장호르몬의 생물학적 활성을 확인하기 위하여, Nb2 세포의 증식 효과를 측정함으로써 활성 분석하였다. In order to confirm the biological activity of purified human growth hormone, activity was analyzed by measuring the proliferative effect of Nb2 cells.

Nb2-11 쥐 림프종 세포는 ECACC(European Collection of Cell Culture)에서 구입하였다. 세포는 RPMI1640배지에(GIB-11875, invitrogen) 2 mM 머캅토에탄올, 세포오염방지 항생제(invitrogen,cat No. 15240-062)및 10% 말 혈청을 첨가하여 배양하였다. 배지는 배양 플라스크 표면에 세포가 80 내지 90% 정도에 이르렀을 때 계대 배양을 실시하였으며, 2 내지 3일을 주기로 교환해 주었다. 배양한 1X104개의 세포를 96웰 플레이트에 3중(triplate)으로 넣은 후, hGH 단백질을 최소 1X10-4 nM 에서 최대 1 nM이 되도록 동일한 배지에 희석하여 웰에 첨가하고 48시간 동안 세포를 자극하였다. Nb2-11 murine lymphoma cells were purchased from the European Collection of Cell Culture (ECACC). Cells were cultured in RPMI1640 medium (GIB-11875, invitrogen) with 2 mM mercaptoethanol, anti-antibacterial antibiotics (invitrogen, cat No. 15240-062) and 10% horse serum. The medium was passaged when the cells reached 80 to 90% of the surface of the culture flask, and exchanged every 2 to 3 days. Cultured 1 × 10 4 cells were triplicated into 96 well plates, then hGH protein was diluted in the same medium to a minimum of 1 × 10 −4 nM to 1 nM, added to the wells and the cells were stimulated for 48 hours. .

각각의 웰의 최종부피는 100㎕로 고정시켰다. 살아있는 세포의 미토콘드리아 탈수소효소의 활성에 의해 노란색의 MTS(Promega, Cell-titer 96 well Aqueous non-radioactive cell proliferation assay kit , Cat No. G5421)가 수용성의 보라색을 띠는 물질로 전환되는 특성을 이용하여 세포 생존률을 측정하였다. The final volume of each well was fixed at 100 μl. By using mitochondrial dehydrogenase activity in living cells, yellow MTS (Promega, Cell-titer 96 well Aqueous non-radioactive cell proliferation assay kit, Cat No. G5421) is converted into water-soluble purple substance. Cell viability was measured.

96웰 플레이트에서 배양한 Nb2 세포에 MTS를 20㎕ 첨가한 후 2시간 동안 37, 5% 이산화탄소 배양기에서 반응시켰다. 최종 반응물은 490 nm에서 흡광도를 측정함으로써 활성을 확인하였다(도 15). 도 15는 hGH의 생물학적 활성확인을 위하여 농도에 따른 Nb2 세포 증식 결과를 보여주는 그래프로서, 도면에 표시된 Bar는 반복실험에 대한 표준편차를 나타낸다. 도 15에서 보듯이, 단백질의 Nb2 세포 증식활성은 시판중인 대조구 인간 성장호르몬 활성과 유사하게 나타나는 것을 확인하였다. 20 μl of MTS was added to Nb2 cells incubated in 96 well plates, and then reacted in 37, 5% carbon dioxide incubator for 2 hours. The final reaction was confirmed for activity by measuring absorbance at 490 nm (FIG. 15). 15 is a graph showing the results of Nb2 cell proliferation according to the concentration for confirming the biological activity of hGH, the bar shown in the figure represents the standard deviation for repeated experiments. As shown in Figure 15, Nb2 cell proliferation activity of the protein was confirmed to appear similar to the commercial control human growth hormone activity.

<110> Korea Research Institute of Bioscience and Biotechnology <120> A novel fabricated translational fusion partner for secretory production of foreign protein, a process for preparing the foreign protein using the same and a screening method of the same <130> PA110444/KR <160> 156 <170> KopatentIn 2.0 <210> 1 <211> 23 <212> PRT <213> TFP1-Pre <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 20 <210> 2 <211> 19 <212> PRT <213> TFP2-Pre <400> 2 Met Thr Pro Tyr Ala Val Ala Ile Thr Val Ala Leu Leu Ile Val Thr 1 5 10 15 Val Ser Ala <210> 3 <211> 21 <212> PRT <213> TFP3-Pre <400> 3 Met Gln Phe Lys Asn Val Ala Leu Ala Ala Ser Val Ala Ala Leu Ser 1 5 10 15 Ala Thr Ala Ser Ala 20 <210> 4 <211> 18 <212> PRT <213> TFP4-Pre <400> 4 Met Arg Phe Ala Glu Phe Leu Val Val Phe Ala Thr Leu Gly Gly Gly 1 5 10 15 Met Ala <210> 5 <211> 24 <212> PRT <213> TFP9-Pre <400> 5 Met Val Phe Gly Gln Leu Tyr Ala Leu Phe Ile Phe Thr Leu Ser Cys 1 5 10 15 Cys Ile Ser Lys Thr Val Gln Ala 20 <210> 6 <211> 19 <212> PRT <213> TFP10-Pre <400> 6 Met Leu Gln Ser Val Val Phe Phe Ala Leu Leu Thr Phe Ala Ser Ser 1 5 10 15 Val Ser Ala <210> 7 <211> 19 <212> PRT <213> TFP11-Pre <400> 7 Met Lys Phe Ser Thr Ala Val Thr Thr Leu Ile Ser Ser Gly Ala Ile 1 5 10 15 Val Ser Ala <210> 8 <211> 22 <212> PRT <213> TFP12-Pre <400> 8 Met Asn Trp Leu Phe Leu Val Ser Leu Val Phe Phe Cys Gly Val Ser 1 5 10 15 Thr His Pro Ala Leu Ala 20 <210> 9 <211> 20 <212> PRT <213> TFP13-Pre <400> 9 Met Lys Phe Ser Ser Val Thr Ala Ile Thr Leu Ala Thr Val Ala Thr 1 5 10 15 Val Ala Thr Ala 20 <210> 10 <211> 20 <212> PRT <213> TFP14-Pre <400> 10 Met Ala Ser Phe Ala Thr Lys Phe Val Ile Ala Cys Phe Leu Phe Phe 1 5 10 15 Ser Ala Ser Ala 20 <210> 11 <211> 18 <212> PRT <213> TFP16-Pre <400> 11 Met Gln Tyr Lys Lys Thr Leu Val Ala Ser Ala Leu Ala Ala Thr Thr 1 5 10 15 Leu Ala <210> 12 <211> 19 <212> PRT <213> TFP17-Pre <400> 12 Met Gln Phe Lys Asn Ala Leu Thr Ala Thr Ala Ile Leu Ser Ala Ser 1 5 10 15 Ala Leu Ala <210> 13 <211> 19 <212> PRT <213> TFP19-Pre <400> 13 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala <210> 14 <211> 19 <212> PRT <213> TFP20-Pre <400> 14 Met Val Ser Lys Thr Trp Ile Cys Gly Phe Ile Ser Ile Ile Thr Val 1 5 10 15 Val Gln Ala <210> 15 <211> 17 <212> PRT <213> TFP21-Pre <400> 15 Met Lys Leu Ser Ala Leu Leu Ala Leu Ser Ala Ser Thr Ala Val Leu 1 5 10 15 Ala <210> 16 <211> 18 <212> PRT <213> TFP22-Pre <400> 16 Met Lys Leu Ser Thr Val Leu Leu Ser Ala Gly Leu Ala Ser Thr Thr 1 5 10 15 Leu Ala <210> 17 <211> 23 <212> PRT <213> TFP23-Pre <400> 17 Met Arg Phe Ser Thr Thr Leu Ala Thr Ala Ala Thr Ala Leu Phe Phe 1 5 10 15 Thr Ala Ser Gln Val Ser Ala 20 <210> 18 <211> 19 <212> PRT <213> TFP24-Pre <400> 18 Met Arg Leu Ser Asn Leu Ile Ala Ser Ala Ser Leu Leu Ser Ala Ala 1 5 10 15 Thr Leu Ala <210> 19 <211> 18 <212> PRT <213> TFP27-Pre <400> 19 Met Gln Phe Ser Thr Val Ala Ser Ile Ala Ala Val Ala Ala Val Ala 1 5 10 15 Ser Ala <210> 20 <211> 18 <212> PRT <213> TFP29-Pre <400> 20 Met Lys Phe Ser Ser Ala Leu Val Leu Ser Ala Val Ala Ala Thr Ala 1 5 10 15 Leu Ala <210> 21 <211> 19 <212> PRT <213> TFP37-Pre <400> 21 Met Lys Phe Gln Val Val Leu Ser Ala Leu Leu Ala Cys Ser Ser Ala 1 5 10 15 Val Val Ala <210> 22 <211> 22 <212> PRT <213> TFP38-Pre <400> 22 Met Arg Ala Ile Thr Leu Leu Ser Ser Val Val Ser Leu Ala Leu Leu 1 5 10 15 Ser Lys Glu Val Leu Ala 20 <210> 23 <211> 20 <212> PRT <213> TFP39-Pre <400> 23 Met Val Lys Leu Thr Ser Ile Val Ala Gly Val Ala Ala Ile Ala Ala 1 5 10 15 Gly Val Ala Ala 20 <210> 24 <211> 18 <212> PRT <213> TFP41-Pre <400> 24 Met Lys Phe Thr Ser Val Leu Ala Phe Phe Leu Ala Thr Leu Thr Ala 1 5 10 15 Ser Ala <210> 25 <211> 23 <212> PRT <213> TFP42-Pre <400> 25 Met Phe Asn Arg Phe Asn Lys Leu Gln Ala Ala Leu Ala Leu Val Leu 1 5 10 15 Tyr Ser Gln Ser Ala Leu Gly 20 <210> 26 <211> 95 <212> PRT <213> TFP1-Pro <400> 26 Asp Ser Tyr Thr Asn Ser Thr Ser Ser Ala Asp Leu Ser Ser Ile Thr 1 5 10 15 Ser Val Ser Ser Ala Ser Ala Ser Ala Thr Ala Ser Asp Ser Leu Ser 20 25 30 Ser Ser Asp Gly Thr Val Tyr Leu Pro Ser Thr Thr Ile Ser Gly Asp 35 40 45 Leu Thr Val Thr Gly Lys Val Ile Ala Thr Glu Ala Val Glu Val Ala 50 55 60 Ala Gly Gly Lys Leu Thr Leu Leu Asp Gly Glu Lys Tyr Val Phe Ser 65 70 75 80 Ser Glu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 85 90 95 <210> 27 <211> 111 <212> PRT <213> TFP2-Pro <400> 27 Leu Gln Val Asn Asn Ser Cys Val Ala Phe Pro Pro Ser Asn Leu Arg 1 5 10 15 Gly Lys Asn Gly Asp Gly Thr Asn Glu Gln Tyr Ala Thr Ala Leu Leu 20 25 30 Ser Ile Pro Trp Asn Gly Pro Pro Glu Ser Ser Arg Asp Ile Asn Leu 35 40 45 Ile Glu Leu Glu Pro Gln Val Ala Leu Tyr Leu Leu Glu Asn Tyr Ile 50 55 60 Asn His Tyr Tyr Asn Thr Thr Arg Asp Asn Lys Cys Pro Asn Asn His 65 70 75 80 Tyr Leu Met Gly Gly Gln Leu Gly Ser Ser Ser Asp Asn Arg Ser Leu 85 90 95 Asn Glu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 100 105 110 <210> 28 <211> 96 <212> PRT <213> TFP3-Pro <400> 28 Glu Gly Tyr Thr Pro Gly Glu Pro Trp Ser Thr Leu Thr Pro Thr Gly 1 5 10 15 Ser Ile Ser Cys Gly Ala Ala Glu Tyr Thr Thr Thr Phe Gly Ile Ala 20 25 30 Val Gln Ala Ile Thr Ser Ser Lys Ala Lys Arg Asp Val Ile Ser Gln 35 40 45 Ile Gly Asp Gly Gln Val Gln Ala Thr Ser Ala Ala Thr Ala Gln Ala 50 55 60 Thr Asp Ser Gln Ala Gln Ala Thr Thr Thr Ala Thr Pro Thr Ser Ser 65 70 75 80 Glu Lys Met Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 85 90 95 <210> 29 <211> 44 <212> PRT <213> TFP4-Pro <400> 29 Ala Pro Val Glu Ser Leu Ala Gly Thr Gln Arg Tyr Leu Val Gln Met 1 5 10 15 Lys Glu Arg Phe Thr Thr Glu Lys Leu Cys Ala Leu Asp Asp Lys Ala 20 25 30 Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 35 40 <210> 30 <211> 202 <212> PRT <213> TFP9-Pro <400> 30 Asp Ser Ser Lys Glu Ser Ser Ser Phe Ile Ser Phe Asp Lys Glu Ser 1 5 10 15 Asn Trp Asp Thr Ile Ser Thr Ile Ser Ser Thr Ala Asp Val Ile Ser 20 25 30 Ser Val Asp Ser Ala Ile Ala Val Phe Glu Phe Asp Asn Phe Ser Leu 35 40 45 Leu Asp Ser Leu Met Ile Asp Glu Glu Tyr Pro Phe Phe Asn Arg Phe 50 55 60 Phe Ala Asn Asp Val Ser Leu Thr Val His Asp Asp Ser Pro Leu Asn 65 70 75 80 Ile Ser Gln Ser Leu Ser Pro Ile Met Glu Gln Phe Thr Val Asp Glu 85 90 95 Leu Pro Glu Ser Ala Ser Asp Leu Leu Tyr Glu Tyr Ser Leu Asp Asp 100 105 110 Lys Ser Ile Val Leu Phe Lys Phe Thr Ser Asp Ala Tyr Asp Leu Lys 115 120 125 Lys Leu Asp Glu Phe Ile Asp Ser Cys Leu Ser Phe Leu Glu Asp Lys 130 135 140 Ser Gly Asp Asn Leu Thr Val Val Ile Asn Ser Leu Gly Trp Ala Phe 145 150 155 160 Glu Asp Glu Asp Gly Asp Asp Glu Tyr Ala Thr Glu Glu Thr Leu Ser 165 170 175 His His Asp Asn Asn Lys Gly Lys Glu Gly Asp Asp Leu Ala Ala Ser 180 185 190 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 195 200 <210> 31 <211> 45 <212> PRT <213> TFP10-Pro <400> 31 Ile Tyr Ser Asn Asn Thr Val Ser Thr Thr Thr Thr Leu Ala Pro Ser 1 5 10 15 Tyr Ser Leu Val Pro Gln Glu Thr Thr Ile Ser Tyr Ala Asp Asp Leu 20 25 30 Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 35 40 45 <210> 32 <211> 119 <212> PRT <213> TFP11-Pro <400> 32 Leu Pro His Val Asp Val His Gln Glu Asp Ala His Gln His Lys Arg 1 5 10 15 Ala Val Ala Tyr Lys Tyr Val Tyr Glu Thr Val Val Val Asp Ser Asp 20 25 30 Gly His Thr Val Thr Pro Ala Ala Ser Glu Val Ala Thr Ala Ala Thr 35 40 45 Ser Ala Ile Ile Thr Thr Ser Val Leu Ala Pro Thr Ser Ser Ala Ala 50 55 60 Ala Ala Asp Ser Ser Ala Ser Ile Ala Val Ser Ser Ala Ala Leu Ala 65 70 75 80 Lys Asn Glu Lys Ile Ser Asp Ala Ala Ala Ser Ala Thr Ala Ser Thr 85 90 95 Ser Gln Gly Ala Ser Ser Ser Ser Tyr Leu Ala Ala Ser Ala Ser Ala 100 105 110 Gly Leu Ala Leu Asp Lys Arg 115 <210> 33 <211> 177 <212> PRT <213> TFP12-Pro <400> 33 Met Ser Ser Asn Arg Leu Leu Lys Leu Ala Asn Lys Ser Pro Lys Lys 1 5 10 15 Ile Ile Pro Leu Lys Asp Ser Ser Phe Glu Asn Ile Leu Ala Pro Pro 20 25 30 His Glu Asn Ala Tyr Ile Val Ala Leu Phe Thr Ala Thr Ala Pro Glu 35 40 45 Ile Gly Cys Ser Leu Cys Leu Glu Leu Glu Ser Glu Tyr Asp Thr Ile 50 55 60 Val Ala Ser Trp Phe Asp Asp His Pro Asp Ala Lys Ser Ser Asn Ser 65 70 75 80 Asp Thr Ser Ile Phe Phe Thr Lys Val Asn Leu Glu Asp Pro Ser Lys 85 90 95 Thr Ile Pro Lys Ala Phe Gln Phe Phe Gln Leu Asn Asn Val Pro Arg 100 105 110 Leu Phe Ile Phe Lys Leu Asn Ser Pro Ser Ile Leu Asp His Ser Val 115 120 125 Ile Ser Ile Ser Thr Asp Thr Gly Ser Glu Arg Met Lys Gln Ile Ile 130 135 140 Gln Ala Ile Lys Gln Phe Ser Gln Val Asn Asp Phe Ser Leu His Leu 145 150 155 160 Pro Val Gly Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys 165 170 175 Arg <210> 34 <211> 57 <212> PRT <213> TFP13-Pro <400> 34 Lys Lys Gly Glu His Asp Phe Thr Thr Thr Leu Thr Leu Ser Ser Asp 1 5 10 15 Gly Ser Leu Thr Thr Thr Thr Ser Thr His Thr Thr His Lys Tyr Gly 20 25 30 Lys Phe Asn Lys Thr Ser Lys Ser Lys Thr Pro Leu Ala Ala Ser Ala 35 40 45 Ser Ala Gly Leu Ala Leu Asp Lys Arg 50 55 <210> 35 <211> 74 <212> PRT <213> TFP14-Pro <400> 35 His Asn Val Leu Leu Pro Ala Tyr Gly Arg Arg Cys Phe Phe Glu Asp 1 5 10 15 Leu Ser Lys Gly Asp Glu Leu Ser Ile Ser Phe Gln Phe Gly Asp Arg 20 25 30 Asn Pro Gln Ser Ser Ser Gln Leu Thr Gly Asp Phe Ile Ile Tyr Gly 35 40 45 Pro Glu Arg His Glu Val Leu Lys Thr Val Arg Glu Leu Ala Ala Ser 50 55 60 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 65 70 <210> 36 <211> 156 <212> PRT <213> TFP16-Pro <400> 36 Ala Tyr Ala Pro Ser Glu Pro Trp Ser Thr Leu Thr Pro Thr Ala Thr 1 5 10 15 Tyr Ser Gly Gly Val Thr Asp Tyr Ala Ser Thr Phe Gly Ile Ala Val 20 25 30 Gln Pro Ile Ser Thr Thr Ser Ser Ala Ser Ser Ala Ala Thr Thr Ala 35 40 45 Ser Ser Lys Ala Lys Arg Ala Ala Ser Gln Ile Gly Asp Gly Gln Val 50 55 60 Gln Ala Ala Thr Thr Thr Ala Ser Val Ser Thr Lys Ser Thr Ala Ala 65 70 75 80 Ala Val Ser Gln Ile Gly Asp Gly Gln Ile Gln Ala Thr Thr Lys Thr 85 90 95 Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Ile Gln Ala Thr 100 105 110 Thr Lys Thr Thr Ser Ala Lys Thr Thr Ala Ala Ala Val Ser Gln Ile 115 120 125 Ser Asp Gly Gln Ile Gln Ala Thr Thr Thr Thr Leu Ala Pro Leu Ala 130 135 140 Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 145 150 155 <210> 37 <211> 190 <212> PRT <213> TFP17-Pro <400> 37 Ala Asn Ser Thr Thr Ser Ile Pro Ser Ser Cys Ser Ile Gly Thr Ser 1 5 10 15 Ala Thr Ala Thr Ala Gln Ala Asp Leu Asp Lys Ile Ser Gly Cys Ser 20 25 30 Thr Ile Val Gly Asn Leu Thr Ile Thr Gly Asp Leu Gly Ser Ala Ala 35 40 45 Leu Ala Ser Ile Gln Glu Ile Asp Gly Ser Leu Thr Ile Phe Asn Ser 50 55 60 Ser Ser Leu Ser Ser Phe Ser Ala Asp Ser Ile Lys Lys Ile Thr Gly 65 70 75 80 Asp Leu Asn Met Gln Glu Leu Ile Ile Leu Thr Ser Ala Ser Phe Gly 85 90 95 Ser Leu Gln Glu Val Asp Ser Ile Asn Met Val Thr Leu Pro Ala Ile 100 105 110 Ser Thr Phe Ser Thr Asp Leu Gln Asn Ala Asn Asn Ile Ile Val Ser 115 120 125 Asp Thr Thr Leu Glu Ser Val Glu Gly Phe Ser Thr Leu Lys Lys Val 130 135 140 Asn Val Phe Asn Ile Asn Asn Asn Arg Tyr Leu Asn Ser Phe Gln Ser 145 150 155 160 Ser Leu Glu Ser Val Ser Asp Ser Leu Gln Phe Ser Ser Asn Gly Asp 165 170 175 Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 180 185 190 <210> 38 <211> 74 <212> PRT <213> TFP19-Pro <400> 38 Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile Pro Ala 1 5 10 15 Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe Asp Val Ala 20 25 30 Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe Ile Asn 35 40 45 Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ala Ala Ser 50 55 60 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 65 70 <210> 39 <211> 138 <212> PRT <213> TFP20-Pro <400> 39 Leu Ser Cys Glu Lys His Asp Val Leu Lys Lys Tyr Gln Val Gly Lys 1 5 10 15 Phe Ser Ser Leu Thr Ser Thr Glu Arg Asp Thr Pro Pro Ser Thr Thr 20 25 30 Ile Glu Lys Trp Trp Ile Asn Val Cys Glu Glu His Asn Val Glu Pro 35 40 45 Pro Glu Glu Cys Lys Lys Asn Asp Met Leu Cys Gly Leu Thr Asp Val 50 55 60 Ile Leu Pro Gly Lys Asp Ala Ile Thr Thr Gln Ile Ile Asp Phe Asp 65 70 75 80 Lys Asn Ile Gly Phe Asn Val Glu Glu Thr Glu Ser Ala Leu Thr Leu 85 90 95 Thr Leu Asn Gly Ala Thr Trp Gly Ala Asn Ser Phe Asp Ala Lys Leu 100 105 110 Glu Phe Gln Cys Asn Asp Asn Met Lys Gln Asp Glu Leu Ala Ala Ser 115 120 125 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 130 135 <210> 40 <211> 81 <212> PRT <213> TFP21-Pro <400> 40 Ala Pro Ala Val His His Ser Asp Asn His His His Asn Asp Lys Arg 1 5 10 15 Ala Val Val Thr Val Thr Gln Tyr Val Asn Ala Asp Gly Ala Val Val 20 25 30 Ile Pro Ala Ala Thr Thr Ala Thr Ser Ala Ala Ala Asp Gly Lys Val 35 40 45 Glu Ser Val Ala Ala Ala Thr Thr Thr Leu Ser Ser Thr Ala Ala Ala 50 55 60 Ala Thr Thr Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys 65 70 75 80 Arg <210> 41 <211> 177 <212> PRT <213> TFP22-Pro <400> 41 Gln Phe Ser Asn Ser Thr Ser Ala Ser Ser Thr Asp Val Thr Ser Ser 1 5 10 15 Ser Ser Ile Ser Thr Ser Ser Gly Ser Val Thr Ile Thr Ser Ser Glu 20 25 30 Ala Pro Glu Ser Asp Asn Gly Thr Ser Thr Ala Ala Pro Thr Glu Thr 35 40 45 Ser Thr Glu Ala Pro Thr Thr Ala Ile Pro Thr Asn Gly Thr Ser Thr 50 55 60 Glu Ala Pro Thr Thr Ala Ile Pro Thr Asn Gly Thr Ser Thr Glu Ala 65 70 75 80 Pro Thr Asp Thr Thr Thr Glu Ala Pro Thr Thr Ala Leu Pro Thr Asn 85 90 95 Gly Thr Ser Thr Glu Ala Pro Thr Asp Thr Thr Thr Glu Ala Pro Thr 100 105 110 Thr Gly Leu Pro Thr Asn Gly Thr Thr Ser Ala Phe Pro Pro Thr Thr 115 120 125 Ser Leu Pro Pro Ser Asn Thr Thr Thr Thr Pro Pro Tyr Asn Pro Ser 130 135 140 Thr Asp Tyr Thr Thr Asp Tyr Thr Val Val Thr Glu Tyr Thr Thr Tyr 145 150 155 160 Cys Pro Glu Arg Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys 165 170 175 Arg <210> 42 <211> 82 <212> PRT <213> TFP23-Pro <400> 42 Ile Gly Glu Leu Ala Phe Asn Leu Gly Val Lys Asn Asn Asp Gly Thr 1 5 10 15 Cys Lys Ser Thr Ser Asp Tyr Glu Thr Glu Leu Gln Ala Leu Lys Ser 20 25 30 Tyr Thr Ser Thr Val Lys Val Tyr Ala Ala Ser Asp Cys Asn Thr Leu 35 40 45 Gln Asn Leu Gly Pro Ala Ala Glu Ala Glu Gly Phe Thr Ile Phe Val 50 55 60 Gly Val Trp Pro Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp 65 70 75 80 Lys Arg <210> 43 <211> 47 <212> PRT <213> TFP24-Pro <400> 43 Ala Pro Ala Asn His Glu His Lys Asp Lys Arg Ala Val Val Thr Thr 1 5 10 15 Thr Val Gln Lys Gln Thr Thr Val Ile Val Asn Gly Ala Ala Ser Thr 20 25 30 Pro Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 35 40 45 <210> 44 <211> 107 <212> PRT <213> TFP27-Pro <400> 44 Ala Ala Asn Val Thr Thr Ala Thr Val Ser Gln Glu Ser Thr Thr Leu 1 5 10 15 Val Thr Ile Thr Ser Cys Glu Asp His Val Cys Ser Glu Thr Val Ser 20 25 30 Pro Ala Leu Val Ser Thr Ala Thr Val Thr Val Asp Asp Val Ile Thr 35 40 45 Gln Tyr Thr Thr Trp Cys Pro Leu Thr Thr Glu Ala Pro Lys Asn Gly 50 55 60 Thr Ser Thr Ala Ala Pro Val Thr Ser Thr Glu Ala Pro Lys Asn Thr 65 70 75 80 Thr Ser Ala Ala Pro Thr His Ser Val Thr Ser Tyr Thr Leu Ala Ala 85 90 95 Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 100 105 <210> 45 <211> 62 <212> PRT <213> TFP29-Pro <400> 45 Glu Ser Ile Thr Thr Thr Ile Thr Ala Thr Lys Asn Gly His Val Tyr 1 5 10 15 Thr Lys Thr Val Thr Gln Asp Ala Thr Phe Val Trp Gly Gly Glu Asp 20 25 30 Ser Tyr Ala Ser Ser Thr Ser Ala Ala Glu Ser Ser Ala Ala Glu Thr 35 40 45 Ser Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 50 55 60 <210> 46 <211> 119 <212> PRT <213> TFP37-Pro <400> 46 Ser Pro Ile Glu Asn Leu Phe Lys Tyr Arg Ala Val Lys Ala Ser His 1 5 10 15 Ser Lys Asn Ile Asn Ser Thr Leu Pro Ala Trp Asn Gly Ser Asn Ser 20 25 30 Ser Asn Val Thr Tyr Ala Asn Gly Thr Asn Ser Thr Thr Asn Thr Thr 35 40 45 Thr Ala Glu Ser Ser Gln Leu Gln Ile Ile Val Thr Gly Gly Gln Val 50 55 60 Pro Ile Thr Asn Ser Ser Leu Thr His Thr Asn Tyr Thr Arg Leu Phe 65 70 75 80 Asn Ser Ser Ser Ala Leu Asn Ile Thr Glu Leu Tyr Asn Val Ala Arg 85 90 95 Val Val Asn Glu Thr Ile Gln Asp Asn Leu Ala Ala Ser Ala Ser Ala 100 105 110 Gly Leu Ala Leu Asp Lys Arg 115 <210> 47 <211> 93 <212> PRT <213> TFP38-Pro <400> 47 Thr Pro Pro Ala Cys Leu Leu Ala Cys Val Ala Gln Val Gly Lys Ser 1 5 10 15 Ser Ser Thr Cys Asp Ser Leu Asn Gln Val Thr Cys Tyr Cys Glu His 20 25 30 Glu Asn Ser Ala Val Lys Lys Cys Leu Asp Ser Ile Cys Pro Asn Asn 35 40 45 Asp Ala Asp Ala Ala Tyr Ser Ala Phe Lys Ser Ser Cys Ser Glu Gln 50 55 60 Asn Ala Ser Leu Gly Asp Ser Ser Gly Ser Ala Ser Ser Ser Val Leu 65 70 75 80 Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 85 90 <210> 48 <211> 113 <212> PRT <213> TFP39-Pro <400> 48 Ala Pro Ala Thr Thr Thr Leu Ser Pro Ser Asp Glu Arg Val Asn Leu 1 5 10 15 Val Glu Leu Gly Val Tyr Val Ser Asp Ile Arg Ala His Leu Ala Glu 20 25 30 Tyr Tyr Met Phe Gln Ala Ala His Pro Thr Glu Thr Tyr Pro Val Glu 35 40 45 Ile Ala Glu Ala Val Phe Asn Tyr Gly Asp Phe Thr Thr Met Leu Thr 50 55 60 Gly Ile Pro Ala Asp Gln Val Thr Arg Val Ile Thr Gly Val Pro Trp 65 70 75 80 Tyr Ser Thr Arg Leu Arg Pro Ala Ile Ser Ser Ala Leu Ser Lys Asp 85 90 95 Gly Ile Tyr Thr Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys 100 105 110 Arg <210> 49 <211> 72 <212> PRT <213> TFP41-Pro <400> 49 Thr Pro Leu Tyr Lys Arg Gln Asn Val Thr Ser Gly Gly Gly Thr Val 1 5 10 15 Pro Val Ile Ile Thr Gly Gly Pro Ala Val Ser Gly Ser Gln Ser Asn 20 25 30 Val Thr Thr Thr Thr Leu Phe Asn Ser Thr Ser Thr Leu Asn Ile Thr 35 40 45 Gln Leu Tyr Gln Ile Ala Thr Gln Val Asn Leu Ala Ala Ser Ala Ser 50 55 60 Ala Gly Leu Ala Leu Asp Lys Arg 65 70 <210> 50 <211> 100 <212> PRT <213> TFP42-Pro <400> 50 Gln Tyr Tyr Thr Asn Ser Ser Ser Ile Ala Ser Asn Ser Ser Thr Ala 1 5 10 15 Val Ser Ser Thr Ser Ser Gly Ser Val Ser Ile Ser Ser Ser Ile Glu 20 25 30 Leu Thr Ser Ser Thr Ser Asp Val Ser Ser Ser Leu Thr Glu Leu Thr 35 40 45 Ser Ser Ser Thr Glu Val Ser Ser Ser Ile Ala Pro Ser Thr Ser Ser 50 55 60 Ser Glu Val Ser Ser Ser Ile Thr Ser Ser Gly Ser Ser Val Ser Gly 65 70 75 80 Ser Ser Ser Ile Thr Ser Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala 85 90 95 Leu Asp Lys Arg 100 <210> 51 <211> 69 <212> DNA <213> TFP1-Pre <400> 51 atgttcaatc gttttaacaa attccaagct gctgtcgctt tggccctact ctctcgcggc 60 gctctcggt 69 <210> 52 <211> 57 <212> DNA <213> TFP2-Pre <400> 52 atgacgccct atgcagtagc aattaccgtg gccttactaa ttgtaacagt gagcgca 57 <210> 53 <211> 63 <212> DNA <213> TFP3-Pre <400> 53 atgcaattca aaaacgtcgc cctagctgcc tccgttgctg ctctatccgc cactgcttct 60 gct 63 <210> 54 <211> 54 <212> DNA <213> TFP4-Pre <400> 54 atgagatttg cagaattctt ggtggtattt gccacgttag gcggggggat ggct 54 <210> 55 <211> 72 <212> DNA <213> TFP9-Pre <400> 55 atggtgttcg gtcagctgta tgcccttttc atcttcacgt tatcatgttg tatttccaaa 60 actgtgcaag ca 72 <210> 56 <211> 69 <212> DNA <213> TFP10-Pre <400> 56 atgttcaatc gttttaacaa attccaagct gctgtcgctt tggccctact ctctcgcggc 60 gctctcggt 69 <210> 57 <211> 57 <212> DNA <213> TFP11-Pre <400> 57 atgaaattct caactgccgt tactacgttg attagttctg gtgccatcgt gtctgct 57 <210> 58 <211> 66 <212> DNA <213> TFP12-Pre <400> 58 atgaattggc tgtttttggt ctcgctggtt ttcttctgcg gcgtgtcaac ccatcctgcc 60 ctggca 66 <210> 59 <211> 60 <212> DNA <213> TFP13-Pre <400> 59 atgaagttct cttctgttac tgctattact ctagccaccg ttgccaccgt tgccactgct 60 60 <210> 60 <211> 60 <212> DNA <213> TFP14-Pre <400> 60 atggcctcat ttgctactaa gtttgtcatt gcttgcttcc tgttcttctc ggcgtccgcc 60 60 <210> 61 <211> 54 <212> DNA <213> TFP16-Pre <400> 61 atgcaataca aaaagacttt ggttgcctct gctttggccg ctactacatt ggcc 54 <210> 62 <211> 57 <212> DNA <213> TFP17-Pre <400> 62 atgcaattca agaacgcttt gactgctact gctattctaa gtgcctccgc tctagct 57 <210> 63 <211> 57 <212> DNA <213> TFP19-Pre <400> 63 atgagatttc cttcaatttt tactgcagtt ttattcgcag catcctccgc attagct 57 <210> 64 <211> 57 <212> DNA <213> TFP20-Pre <400> 64 atggtatcga agacttggat atgtggcttc atcagtataa ttacagtggt acaggcc 57 <210> 65 <211> 51 <212> DNA <213> TFP21-Pre <400> 65 atgaaattat ccgctctatt agctttatca gcctccaccg ccgtcttggc c 51 <210> 66 <211> 54 <212> DNA <213> TFP22-Pre <400> 66 atgaaattat caactgtcct attatctgcc ggtttagcct cgactacttt ggcc 54 <210> 67 <211> 69 <212> DNA <213> TFP23-Pre <400> 67 atgcgtttct ctactacact cgctactgca gctactgcgc tatttttcac agcctcccaa 60 gtttcagct 69 <210> 68 <211> 57 <212> DNA <213> TFP24-Pre <400> 68 atgcgtctct ctaacctaat tgcttctgcc tctcttttat ctgctgctac tcttgct 57 <210> 69 <211> 54 <212> DNA <213> TFP27-Pre <400> 69 atgcaatttt ctactgtcgc ttctatcgcc gctgtcgccg ctgtcgcttc tgcc 54 <210> 70 <211> 54 <212> DNA <213> TFP29-Pre <400> 70 atgaaattct cttccgcttt ggttctatct gctgttgccg ctactgctct tgct 54 <210> 71 <211> 57 <212> DNA <213> TFP37-Pre <400> 71 atgaagttcc aagttgtttt atctgccctt ttggcatgtt catctgccgt cgtcgca 57 <210> 72 <211> 66 <212> DNA <213> TFP38-Pre <400> 72 atgcgtgcca tcactttatt atcttcagtc gtttctttgg cattgttgtc gaaggaagtc 60 ttagca 66 <210> 73 <211> 60 <212> DNA <213> TFP39-Pre <400> 73 atggtcaaac taacttcaat tgttgctggt gtcgctgcta ttgctgctgg tgtcgctgct 60 60 <210> 74 <211> 54 <212> DNA <213> TFP41-Pre <400> 74 atgaaattca catcagtgct agcatttttt cttgcaactt taacagcttc tgca 54 <210> 75 <211> 69 <212> DNA <213> TFP42-Pre <400> 75 atgttcaatc gctttaataa acttcaagcc gctttggctt tggtccttta ctcccaaagt 60 gcattgggc 69 <210> 76 <211> 285 <212> DNA <213> TFP1-Pro <400> 76 gactcttaca ccaatagcac ctcctccgca gacttgagtt ctatcacttc cgtctcgtca 60 gctagtgcaa gtgccaccgc ttccgactca ctttcttcca gtgacggtac cgtttatttg 120 ccatccacaa caattagcgg tgatctcaca gttactggta aagtaattgc aaccgaggcc 180 gtggaagtcg ctgccggtgg taagttgact ttacttgacg gtgaaaaata cgtcttctca 240 tctgaggccg cctcggcctc tgctggcctc gccttagata aaaga 285 <210> 77 <211> 333 <212> DNA <213> TFP2-Pro <400> 77 ctccaggtca acaattcatg tgtcgctttt ccgccatcaa atctcagggg caagaatgga 60 gacggtacta atgaacagta tgcaactgca ctactttcta ttccctggaa tgggcctcct 120 gagtcatcga gggatattaa tcttatcgaa ctcgaaccgc aagttgcact ctatttgctc 180 gaaaattata ttaaccatta ctacaacacc acaagagaca ataagtgccc taataaccac 240 tacctaatgg gagggcagtt gggtagctca tcggataata ggagtttgaa cgaggccgcc 300 tcggcctctg ctggcctcgc cttagataaa aga 333 <210> 78 <211> 288 <212> DNA <213> TFP3-Pro <400> 78 gaaggttaca ctccaggtga accatggtcc accttaaccc caaccggctc catctcttgt 60 ggtgcagccg aatacactac cacctttggt attgctgttc aagctattac ctcttcaaaa 120 gctaagagag acgttatctc tcaaattggt gacggtcaag tccaagccac ttctgctgct 180 actgctcaag ccaccgatag tcaagcccaa gctactacta ccgctacccc aaccagctcc 240 gaaaagatgg ccgcctcggc ctctgctggc ctcgccttag ataaaaga 288 <210> 79 <211> 132 <212> DNA <213> TFP4-Pro <400> 79 gcaccggttg agtctctggc cgggacccaa cggtatctgg tgcaaatgaa ggagcggttc 60 accacagaga agctgtgtgc tttggacgac aaggccgcct cggcctctgc tggcctcgcc 120 ttagataaaa ga 132 <210> 80 <211> 606 <212> DNA <213> TFP9-Pro <400> 80 gattcatcca aggaaagctc ttcctttatt tcgttcgaca aagagagtaa ctgggatacc 60 atcagcacta tatcttcaac ggcagatgtt atatcatccg ttgacagtgc tatcgctgtt 120 tttgaatttg acaatttctc attattggac agcttgatga ttgacgaaga atacccattc 180 ttcaatagat tctttgccaa tgatgtcagt ttaactgttc atgacgattc gcctttgaac 240 atctctcaat cattatctcc cattatggaa caatttactg tggatgaatt acctgaaagt 300 gcctctgact tactatatga atactcctta gatgataaaa gcatcgtttt gttcaagttt 360 acctcggatg cctacgattt gaaaaaatta gatgaattta ttgattcttg cttatcgttt 420 ttggaagata aatctggcga caatttgact gtggttatta actctcttgg ttgggctttt 480 gaagatgaag atggtgacga tgaatatgca acagaagaga ctttgagcca tcatgataac 540 aacaagggta aagaaggcga cgatctggcc gcctcggcct ctgctggcct cgccttagat 600 aaaaga 606 <210> 81 <211> 135 <212> DNA <213> TFP10-Pro <400> 81 atttattcaa acaatactgt ttctacaact accactttag cgcccagcta ctccttggtg 60 ccccaagaga ctaccatatc gtacgccgac gacctggccg cctcggcctc tgctggcctc 120 gccttagata aaaga 135 <210> 82 <211> 357 <212> DNA <213> TFP11-Pro <400> 82 ttaccacacg tggatgttca ccaagaagat gcccaccaac ataagagggc cgttgcgtac 60 aaatacgttt acgaaactgt tgttgtcgat tctgatggcc acactgtaac tcctgctgct 120 tcagaagtcg ctactgctgc tacctctgct atcattacaa catctgtgtt ggctccaacc 180 tcctccgcag ccgctgcgga tagctccgct tccattgctg tttcatctgc tgccttagcc 240 aagaatgaga aaatctctga tgccgctgca tctgccactg cctcaacatc tcaaggggca 300 tcctcctcat cctacctggc cgcctcggcc tctgctggcc tcgccttaga taaaaga 357 <210> 83 <211> 531 <212> DNA <213> TFP12-Pro <400> 83 atgtccagca acagactact aaagctggct aataaatctc ccaagaaaat tatacctctg 60 aaggactcaa gttttgaaaa catcttggca ccacctcacg aaaatgccta tatagttgct 120 ctgtttactg ccacagcgcc cgaaattggc tgttctctgt gtctcgagct agaatccgaa 180 tacgacacca tagtggcctc ctggtttgat gatcatccgg atgcaaaatc gtccaattcc 240 gatacatcta ttttcttcac aaaggtcaat ttggaggacc cttctaagac cattcctaaa 300 gcgttccagt ttttccaact aaacaatgtt cctagattgt tcatcttcaa actaaactct 360 ccctctattc tggaccacag cgtgatcagt atttccactg atactggctc agaaagaatg 420 aagcaaatca tacaagccat taagcagttc tcgcaagtaa acgacttctc tttacactta 480 cctgtgggtc tggccgcctc ggcctctgct ggcctcgcct tagataaaag a 531 <210> 84 <211> 171 <212> DNA <213> TFP13-Pro <400> 84 aagaagggtg aacatgattt cactaccact ttaactttgt catcggacgg tagtttaact 60 actaccacct ctactcatac cactcacaag tatggtaagt tcaacaagac ttccaagtcc 120 aagacccccc tggccgcctc ggcctctgct ggcctcgcct tagataaaag a 171 <210> 85 <211> 222 <212> DNA <213> TFP14-Pro <400> 85 cataatgtcc ttcttccagc ttatggccgt agatgcttct tcgaagactt gagtaagggt 60 gacgagctct ccatttcgtt ccagttcggt gatagaaacc ctcaatccag tagccagctg 120 actggtgact ttatcatcta cgggccggaa agacatgaag ttttgaaaac ggttagggaa 180 ctggccgcct cggcctctgc tggcctcgcc ttagataaaa ga 222 <210> 86 <211> 468 <212> DNA <213> TFP16-Pro <400> 86 gcctatgctc catctgagcc ttggtccact ttgactccaa cagccactta cagcggtggt 60 gttaccgact acgcttccac cttcggtatt gccgttcaac caatctccac tacatccagc 120 gcatcatctg cagccaccac agcctcatct aaggccaaga gagctgcttc ccaaattggt 180 gatggtcaag tccaagctgc taccactact gcttctgtct ctaccaagag taccgctgcc 240 gccgtttctc agatcggtga tggtcaaatc caagctacta ccaagactac cgctgctgct 300 gtctctcaaa ttggtgatgg tcaaattcaa gctaccacca agactacctc tgctaagact 360 accgccgctg ccgtttctca aatcagtgat ggtcaaatcc aagctaccac cactacttta 420 gcccctctgg ccgcctcggc ctctgctggc ctcgccttag ataaaaga 468 <210> 87 <211> 570 <212> DNA <213> TFP17-Pro <400> 87 gctaactcaa ctacttctat tccatcttca tgtagtattg gtacttctgc cactgctact 60 gctcaagctg atttggacaa aatctccggt tgtagtacca ttgttggtaa cttgaccatc 120 accggtgact tgggttccgc tgctttggct agtatccaag agattgatgg ttccttgact 180 atcttcaact ccagttcttt atcttctttc tccgctgact ctatcaagaa aatcaccggt 240 gatttgaaca tgcaagaatt gatcattttg accagtgctt ctttcggttc tttgcaagaa 300 gtagactcca ttaacatggt gactttgcct gccatttcta ccttctccac cgatttacaa 360 aatgctaaca acattattgt ttctgacacc actttggaaa gtgtcgaagg tttctccact 420 ttgaagaagg ttaatgtttt taacatcaac aacaacagat atctaaactc tttccaatct 480 tccttggaaa gtgtctctga ctctttacaa ttctcttcca acggtgacct ggccgcctcg 540 gcctctgctg gcctcgcctt agataaaaga 570 <210> 88 <211> 222 <212> DNA <213> TFP19-Pro <400> 88 gctccagtca acactacaac agaagatgaa acggcacaaa ttccggctga agctgtcatc 60 ggttacttag atttagaagg ggatttcgat gttgctgttt tgccattttc caacagcaca 120 aataacgggt tattgtttat aaatactact attgccagca ttgctgctaa agaagaaggg 180 gtggccgcct cggcctctgc tggcctcgcc ttagataaaa ga 222 <210> 89 <211> 414 <212> DNA <213> TFP20-Pro <400> 89 ttgtcctgcg agaagcatga tgtattgaaa aagtatcagg tgggaaaatt tagctcacta 60 acttctacgg aaagggatac tccgccaagc acaactattg aaaagtggtg gataaacgtt 120 tgcgaagagc ataacgtaga acctcctgaa gaatgtaaaa aaaatgacat gctatgtggt 180 ttaacagatg tcatcttgcc cggtaaggat gctatcacca ctcaaattat agattttgac 240 aaaaacattg gcttcaatgt cgaggaaact gagagtgcgc ttacattgac actaaacggc 300 gctacgtggg gcgccaattc ttttgacgca aaactagaat ttcagtgtaa tgacaatatg 360 aaacaagacg aactggccgc ctcggcctct gctggcctcg ccttagataa aaga 414 <210> 90 <211> 243 <212> DNA <213> TFP21-Pro <400> 90 gctccagctg tccaccatag tgacaaccac caccacaacg acaagcgtgc cgttgtcacc 60 gttactcagt acgtcaacgc agacggcgct gttgttattc cagctgccac caccgctacc 120 tcggcggctg ctgatggaaa ggtcgagtct gttgctgctg ccaccactac tttgtcctcg 180 actgccgccg ccgctacaac cctggccgcc tcggcctctg ctggcctcgc cttagataaa 240 aga 243 <210> 91 <211> 531 <212> DNA <213> TFP22-Pro <400> 91 caattttcca acagtacatc tgcttcttcc accgatgtca cttcctcctc ttccatctcc 60 acttcctctg gctcagtaac tatcacatct tctgaagctc cagaatccga caacggtacc 120 agcacagctg caccaactga aacctcaaca gaggctccaa ccactgctat cccaactaac 180 ggtacctcta ctgaagctcc aaccactgct atcccaacta acggtacctc tactgaagct 240 ccaactgata ctactactga agctccaacc accgctcttc caactaacgg tacttctact 300 gaagctccaa ctgatactac tactgaagct ccaaccaccg gtcttccaac caacggtacc 360 acttcagctt tcccaccaac tacatctttg ccaccaagca acactaccac cactcctcct 420 tacaacccat ctactgacta caccactgac tacactgtag tcactgaata tactacttac 480 tgtccggaac gggccgcctc ggcctctgct ggcctcgcct tagataaaag a 531 <210> 92 <211> 246 <212> DNA <213> TFP23-Pro <400> 92 attggtgaac tagcctttaa cttgggtgtc aagaacaacg atggtacttg taagtccact 60 tccgactatg aaaccgaatt acaagctttg aagagctaca cttccaccgt caaagtttac 120 gctgcctcag attgtaacac tttgcaaaac ttaggtcctg ctgctgaagc tgagggattt 180 actatctttg tcggtgtttg gccactggcc gcctcggcct ctgctggcct cgccttagat 240 aaaaga 246 <210> 93 <211> 141 <212> DNA <213> TFP24-Pro <400> 93 gcccccgcta accacgaaca caaggacaag cgtgctgtgg tcactaccac tgttcaaaaa 60 caaaccactg tcattgttaa tggtgccgct tcaactcccc tggccgcctc ggcctctgct 120 ggcctcgcct tagataaaag a 141 <210> 94 <211> 321 <212> DNA <213> TFP27-Pro <400> 94 gctgctaacg ttaccactgc tactgtcagc caagaatcta ccactttggt caccatcact 60 tcttgtgaag accacgtctg ttctgaaact gtctccccag ctttggtttc caccgctacc 120 gtcaccgtcg atgacgttat cactcaatac accacctggt gcccattgac cactgaagcc 180 ccaaagaacg gtacttctac tgctgctcca gttacctcta ctgaagctcc aaagaacacc 240 acctctgctg ctccaactca ctctgtcacc tcttacacgc tggccgcctc ggcctctgct 300 ggcctcgcct tagataaaag a 321 <210> 95 <211> 186 <212> DNA <213> TFP29-Pro <400> 95 gagagtatca ccaccaccat cactgccacc aagaacggtc atgtctacac taagactgtc 60 acccaagatg ctacttttgt ttggggtggt gaagactctt acgccagcag cacttctgcc 120 gctgaatctt ctgccgccga aacttcggcc gcctcggcct ctgctggcct cgccttagat 180 aaaaga 186 <210> 96 <211> 357 <212> DNA <213> TFP37-Pro <400> 96 agcccaatcg aaaacctatt caaatacagg gcagttaagg catctcacag taagaatatc 60 aactccactt tgccggcctg gaatgggtct aactctagca atgttaccta cgctaatgga 120 acaaacagta ctaccaatac tactactgcc gaaagcagtc aattacaaat cattgtaaca 180 ggtggtcaag taccaatcac caacagttct ttgacccaca caaactacac cagattattc 240 aacagttctt ctgctttgaa cattaccgaa ttgtacaatg ttgcccgtgt tgttaacgaa 300 acgatccaag ataacctggc cgcctcggcc tctgctggcc tcgccttaga taaaaga 357 <210> 97 <211> 279 <212> DNA <213> TFP38-Pro <400> 97 acacctccag cttgtttatt ggcctgtgtt gcgcaagtcg gcaaatcctc ttccacatgt 60 gactctttga atcaagtcac ctgttactgt gaacacgaaa actccgccgt caagaaatgt 120 ctagactcca tctgcccaaa caatgacgct gatgctgctt attctgcttt caagagttct 180 tgttccgaac aaaatgcttc attgggcgat tccagcggca gtgcctcctc atccgttctg 240 gccgcctcgg cctctgctgg cctcgcctta gataaaaga 279 <210> 98 <211> 339 <212> DNA <213> TFP39-Pro <400> 98 gccccagcca ccactacttt atctccctct gatgaaagag ttaacctggt cgaattaggt 60 gtctacgtct cagatatcag agctcatttg gctgaatact atatgttcca agctgctcat 120 ccaactgaaa cttacccagt tgaaattgct gaagctgttt tcaactacgg tgatttcacc 180 actatgttga ctggtattcc cgctgatcaa gtcactagag tcatcactgg tgtcccatgg 240 tactccacca gattgagacc agctatctcc agcgctctat ccaaggacgg tatctacacg 300 gccgcctcgg cctctgctgg cctcgcctta gataaaaga 339 <210> 99 <211> 216 <212> DNA <213> TFP41-Pro <400> 99 acaccacttt acaagaggca gaacgttact tctggcggcg gtacggtccc cgtgatcatc 60 acgggtggac ctgctgtatc tggtagccag tcaaacgtta ctaccacaac gctattcaac 120 tctacttcca ccttaaacat cactcaactt taccaaattg ctactcaagt taatctggcc 180 gcctcggcct ctgctggcct cgccttagat aaaaga 216 <210> 100 <211> 300 <212> DNA <213> TFP42-Pro <400> 100 caatattata ccaacagttc ctcaatcgct agtaacagct ccaccgccgt ttcgtcaact 60 tcatcaggtt ccgtttccat cagtagttct attgagttga cctcatctac ttctgatgtc 120 tcgagctctc tcactgagtt aacgtcatcc tccaccgaag tctcgagctc cattgctcca 180 tcaacctcgt cctctgaagt ctcgagctct attacttcat caggctcttc agtctccggc 240 tcatcttcta ttacttccct ggccgcctcg gcctctgctg gcctcgcctt agataaaaga 300 300 <210> 101 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer GAL100 <400> 101 gtatatggtg gtaatgccat g 21 <210> 102 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T1-H170 <400> 102 accgagagcg ccgcgagaga g 21 <210> 103 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T2-H171 <400> 103 tgcgctcact gttacaatta g 21 <210> 104 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T3-H172 <400> 104 agcagaagca gtggcggata g 21 <210> 105 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T4-H173 <400> 105 agccatcccc ccgcctaacg tg 22 <210> 106 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T9-H174 <400> 106 tgcttgcaca gttttggaaa tac 23 <210> 107 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T10-H175 <400> 107 cgctgacaca gaacttgcg 19 <210> 108 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T11-H176 <400> 108 agcagacacg atggcaccag 20 <210> 109 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T12-H177 <400> 109 tgccagggca ggatgggttg 20 <210> 110 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T13-H178 <400> 110 agcagtggca acggtggcaa c 21 <210> 111 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T14-H179 <400> 111 ggcggacgcc gagaagaac 19 <210> 112 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T16-H180 <400> 112 ggccaatgta gtagcggcc 19 <210> 113 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T17-H181 <400> 113 agctagagcg gaggcactta g 21 <210> 114 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T19-H182 <400> 114 agctaatgcg gaggatgctg cg 22 <210> 115 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T20-H183 <400> 115 ggcctgtacc actgtaatta t 21 <210> 116 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T21-H184 <400> 116 ggccaagacg gcggtggag 19 <210> 117 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T22-H185 <400> 117 ggccaaagta gtcgaggct 19 <210> 118 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T23-H186 <400> 118 agctgaaact tgggaggctg 20 <210> 119 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer T24-H187 <400> 119 agcaagagta gcagcagata aaag 24 <210> 120 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T27-H188 <400> 120 ggcagaagcg acagcggcg 19 <210> 121 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T29-H189 <400> 121 agcaagagca gtagcggca 19 <210> 122 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T37-H190 <400> 122 tgcgacgacg gcagatgaac 20 <210> 123 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T38-H191 <400> 123 tgctaagact tccttcgaca ac 22 <210> 124 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T39-H192 <400> 124 agcagcgaca ccagcagca 19 <210> 125 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T41-H193 <400> 125 tgcagaagct gttaaagttg 20 <210> 126 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T42-H194 <400> 126 gcccaatgca ctttgggagt 20 <210> 127 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T1-H225 <400> 127 gactcttaca ccaatagcac 20 <210> 128 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T2-H226 <400> 128 ctccaggtca acaattcatg tg 22 <210> 129 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T3-H227 <400> 129 gaaggttaca ctccaggtga ac 22 <210> 130 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T4-H228 <400> 130 gcaccggttg agtctctggc 20 <210> 131 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T9-H229 <400> 131 gattcatcca aggaaagctc ttc 23 <210> 132 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T10-H230 <400> 132 atttattcaa acaatactgt ttc 23 <210> 133 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T11-H231 <400> 133 ttaccacacg tggatgttca c 21 <210> 134 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer T12-H232 <400> 134 atgtccagca acagactact aaag 24 <210> 135 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T13-H233 <400> 135 aagaagggtg aacatgattt cac 23 <210> 136 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T14-H234 <400> 136 cataatgtcc ttcttccagc 20 <210> 137 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T16-H235 <400> 137 gcctatgctc catctgagcc ttg 23 <210> 138 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T17-H236 <400> 138 gctaactcaa ctacttctat tc 22 <210> 139 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T19-H237 <400> 139 gctccagtca acactacaac 20 <210> 140 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T20-H238 <400> 140 ttgtcctgcg agaagcatga tg 22 <210> 141 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T21-H239 <400> 141 gctccagctg tccaccatag tg 22 <210> 142 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T22-H240 <400> 142 caattttcca acagtacatc tg 22 <210> 143 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T23-H241 <400> 143 attggtgaac tagcctttaa c 21 <210> 144 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T24-H242 <400> 144 gctcccgcta accacgaaca c 21 <210> 145 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T27-H243 <400> 145 gctgctaacg ttaccactgc tac 23 <210> 146 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T29-H244 <400> 146 gagagtatca ccaccaccat c 21 <210> 147 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T37-H245 <400> 147 agcccaatcg aaaacctatt c 21 <210> 148 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T38-H246 <400> 148 acacctccag cttgtttatt gg 22 <210> 149 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T39-H247 <400> 149 gccccagcca ccactacttt atc 23 <210> 150 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T41-H248 <400> 150 acaccacttt acaagaggca g 21 <210> 151 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T42-H249 <400> 151 caatattata ccaacagttc ctc 23 <210> 152 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer GT90R <400> 152 ggaaaggacc actcttacat aactag 26 <210> 153 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> primer GT70r <400> 153 tcagatttac agataatgat gtcattatta aatatatata tatatatatt gtcactccgt 60 tcaagtcgac 70 <210> 154 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer SH32 <400> 154 ctcgccttag ataaaagatt cccaaccatt cccttatc 38 <210> 155 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer SH33 <400> 155 cactccgttc aagtcgacct agaagccaca gctgccct 38 <210> 156 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer LNK39 <400> 156 ggccgcctcg gcctctgctg gcctcgcctt agataaaaga 40 <110> Korea Research Institute of Bioscience and Biotechnology <120> A novel fabricated translational fusion partner for secretory          production of foreign protein, a process for preparing the          foreign protein using the same and a screening method of the same <130> PA110444 / KR <160> 156 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> PRT <213> TFP1-Pre <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              20 <210> 2 <211> 19 <212> PRT <213> TFP2-Pre <400> 2 Met Thr Pro Tyr Ala Val Ala Ile Thr Val Ala Leu Leu Ile Val Thr   1 5 10 15 Val Ser Ala             <210> 3 <211> 21 <212> PRT <213> TFP3-Pre <400> 3 Met Gln Phe Lys Asn Val Ala Leu Ala Ala Ser Val Ala Ala Leu Ser   1 5 10 15 Ala Thr Ala Ser Ala              20 <210> 4 <211> 18 <212> PRT <213> TFP4-Pre <400> 4 Met Arg Phe Ala Glu Phe Leu Val Val Phe Ala Thr Leu Gly Gly Gly   1 5 10 15 Met ala         <210> 5 <211> 24 <212> PRT <213> TFP9-Pre <400> 5 Met Val Phe Gly Gln Leu Tyr Ala Leu Phe Ile Phe Thr Leu Ser Cys   1 5 10 15 Cys Ile Ser Lys Thr Val Gln Ala              20 <210> 6 <211> 19 <212> PRT <213> TFP10-Pre <400> 6 Met Leu Gln Ser Val Val Phe Phe Ala Leu Leu Thr Phe Ala Ser Ser   1 5 10 15 Val Ser Ala             <210> 7 <211> 19 <212> PRT <213> TFP11-Pre <400> 7 Met Lys Phe Ser Thr Ala Val Thr Thr Leu Ile Ser Ser Gly Ala Ile   1 5 10 15 Val Ser Ala             <210> 8 <211> 22 <212> PRT <213> TFP12-Pre <400> 8 Met Asn Trp Leu Phe Leu Val Ser Leu Val Phe Phe Cys Gly Val Ser   1 5 10 15 Thr His Pro Ala Leu Ala              20 <210> 9 <211> 20 <212> PRT <213> TFP13-Pre <400> 9 Met Lys Phe Ser Ser Val Thr Ala Ile Thr Leu Ala Thr Val Ala Thr   1 5 10 15 Val Ala Thr Ala              20 <210> 10 <211> 20 <212> PRT <213> TFP14-Pre <400> 10 Met Ala Ser Phe Ala Thr Lys Phe Val Ile Ala Cys Phe Leu Phe Phe   1 5 10 15 Ser Ala Ser Ala              20 <210> 11 <211> 18 <212> PRT <213> TFP16-Pre <400> 11 Met Gln Tyr Lys Lys Thr Leu Val Ala Ser Ala Leu Ala Ala Thr Thr   1 5 10 15 Leu Ala         <210> 12 <211> 19 <212> PRT <213> TFP17-Pre <400> 12 Met Gln Phe Lys Asn Ala Leu Thr Ala Thr Ala Ile Leu Ser Ala Ser   1 5 10 15 Ala Leu Ala             <210> 13 <211> 19 <212> PRT <213> TFP19-Pre <400> 13 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser   1 5 10 15 Ala Leu Ala             <210> 14 <211> 19 <212> PRT <213> TFP20-Pre <400> 14 Met Val Ser Lys Thr Trp Ile Cys Gly Phe Ile Ser Ile Ile Thr Val   1 5 10 15 Val Gln Ala             <210> 15 <211> 17 <212> PRT <213> TFP21-Pre <400> 15 Met Lys Leu Ser Ala Leu Leu Ala Leu Ser Ala Ser Thr Ala Val Leu   1 5 10 15 Ala     <210> 16 <211> 18 <212> PRT <213> TFP22-Pre <400> 16 Met Lys Leu Ser Thr Val Leu Leu Ser Ala Gly Leu Ala Ser Thr Thr   1 5 10 15 Leu Ala         <210> 17 <211> 23 <212> PRT <213> TFP23-Pre <400> 17 Met Arg Phe Ser Thr Thr Leu Ala Thr Ala Ala Thr Ala Leu Phe Phe   1 5 10 15 Thr Ala Ser Gln Val Ser Ala              20 <210> 18 <211> 19 <212> PRT <213> TFP24-Pre <400> 18 Met Arg Leu Ser Asn Leu Ile Ala Ser Ala Ser Leu Leu Ser Ala Ala   1 5 10 15 Thr leu ala             <210> 19 <211> 18 <212> PRT <213> TFP27-Pre <400> 19 Met Gln Phe Ser Thr Val Ala Ser Ile Ala Ala Val Ala Ala Val Ala   1 5 10 15 Ser ala         <210> 20 <211> 18 <212> PRT <213> TFP29-Pre <400> 20 Met Lys Phe Ser Ser Ala Leu Val Leu Ser Ala Val Ala Ala Thr Ala   1 5 10 15 Leu Ala         <210> 21 <211> 19 <212> PRT <213> TFP37-Pre <400> 21 Met Lys Phe Gln Val Val Leu Ser Ala Leu Leu Ala Cys Ser Ser Ala   1 5 10 15 Val Val Ala             <210> 22 <211> 22 <212> PRT <213> TFP38-Pre <400> 22 Met Arg Ala Ile Thr Leu Leu Ser Ser Val Val Ser Leu Ala Leu Leu   1 5 10 15 Ser Lys Glu Val Leu Ala              20 <210> 23 <211> 20 <212> PRT <213> TFP39-Pre <400> 23 Met Val Lys Leu Thr Ser Ile Val Ala Gly Val Ala Ala Ile Ala Ala   1 5 10 15 Gly val ala ala              20 <210> 24 <211> 18 <212> PRT <213> TFP41-Pre <400> 24 Met Lys Phe Thr Ser Val Leu Ala Phe Phe Leu Ala Thr Leu Thr Ala   1 5 10 15 Ser ala         <210> 25 <211> 23 <212> PRT <213> TFP42-Pre <400> 25 Met Phe Asn Arg Phe Asn Lys Leu Gln Ala Ala Leu Ala Leu Val Leu   1 5 10 15 Tyr Ser Gln Ser Ala Leu Gly              20 <210> 26 <211> 95 <212> PRT <213> TFP1-Pro <400> 26 Asp Ser Tyr Thr Asn Ser Thr Ser Ser Ala Asp Leu Ser Ser Ile Thr   1 5 10 15 Ser Val Ser Ser Ala Ser Ala Ser Ala Thr Ala Ser Asp Ser Leu Ser              20 25 30 Ser Ser Asp Gly Thr Val Tyr Leu Pro Ser Thr Thr Ile Ser Gly Asp          35 40 45 Leu Thr Val Thr Gly Lys Val Ile Ala Thr Glu Ala Val Glu Val Ala      50 55 60 Ala Gly Gly Lys Leu Thr Leu Leu Asp Gly Glu Lys Tyr Val Phe Ser  65 70 75 80 Ser Glu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg                  85 90 95 <210> 27 <211> 111 <212> PRT <213> TFP2-Pro <400> 27 Leu Gln Val Asn Asn Ser Cys Val Ala Phe Pro Pro Ser Asn Leu Arg   1 5 10 15 Gly Lys Asn Gly Asp Gly Thr Asn Glu Gln Tyr Ala Thr Ala Leu Leu              20 25 30 Ser Ile Pro Trp Asn Gly Pro Pro Glu Ser Ser Arg Asp Ile Asn Leu          35 40 45 Ile Glu Leu Glu Pro Gln Val Ala Leu Tyr Leu Leu Glu Asn Tyr Ile      50 55 60 Asn His Tyr Tyr Asn Thr Thr Arg Asp Asn Lys Cys Pro Asn Asn His  65 70 75 80 Tyr Leu Met Gly Gly Gln Leu Gly Ser Ser Ser Ser Asp Asn Arg Ser Leu                  85 90 95 Asn Glu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg             100 105 110 <210> 28 <211> 96 <212> PRT <213> TFP3-Pro <400> 28 Glu Gly Tyr Thr Pro Gly Glu Pro Trp Ser Thr Leu Thr Pro Thr Gly   1 5 10 15 Ser Ile Ser Cys Gly Ala Ala Glu Tyr Thr Thr Thr Phe Gly Ile Ala              20 25 30 Val Gln Ala Ile Thr Ser Ser Lys Ala Lys Arg Asp Val Ile Ser Gln          35 40 45 Ile Gly Asp Gly Gln Val Gln Ala Thr Ser Ala Ala Thr Ala Gln Ala      50 55 60 Thr Asp Ser Gln Ala Gln Ala Thr Thr Thr Thr Ala Thr Pro Thr Ser Ser  65 70 75 80 Glu Lys Met Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg                  85 90 95 <210> 29 <211> 44 <212> PRT <213> TFP4-Pro <400> 29 Ala Pro Val Glu Ser Leu Ala Gly Thr Gln Arg Tyr Leu Val Gln Met   1 5 10 15 Lys Glu Arg Phe Thr Thr Glu Lys Leu Cys Ala Leu Asp Asp Lys Ala              20 25 30 Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg          35 40 <210> 30 <211> 202 <212> PRT <213> TFP9-Pro <400> 30 Asp Ser Ser Lys Glu Ser Ser Ser Phe Ile Ser Phe Asp Lys Glu Ser   1 5 10 15 Asn Trp Asp Thr Ile Ser Thr Ile Ser Ser Thr Ala Asp Val Ile Ser              20 25 30 Ser Val Asp Ser Ala Ile Ala Val Phe Glu Phe Asp Asn Phe Ser Leu          35 40 45 Leu Asp Ser Leu Met Ile Asp Glu Glu Tyr Pro Phe Phe Asn Arg Phe      50 55 60 Phe Ala Asn Asp Val Ser Leu Thr Val His Asp Asp Ser Pro Leu Asn  65 70 75 80 Ile Ser Gln Ser Leu Ser Pro Ile Met Glu Gln Phe Thr Val Asp Glu                  85 90 95 Leu Pro Glu Ser Ala Ser Asp Leu Leu Tyr Glu Tyr Ser Leu Asp Asp             100 105 110 Lys Ser Ile Val Leu Phe Lys Phe Thr Ser Asp Ala Tyr Asp Leu Lys         115 120 125 Lys Leu Asp Glu Phe Ile Asp Ser Cys Leu Ser Phe Leu Glu Asp Lys     130 135 140 Ser Gly Asp Asn Leu Thr Val Val Ile Asn Ser Leu Gly Trp Ala Phe 145 150 155 160 Glu Asp Glu Asp Gly Asp Asp Glu Tyr Ala Thr Glu Glu Thr Leu Ser                 165 170 175 His His Asp Asn Asn Lys Gly Lys Glu Gly Asp Asp Leu Ala Ala Ser             180 185 190 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg         195 200 <210> 31 <211> 45 <212> PRT <213> TFP10-Pro <400> 31 Ile Tyr Ser Asn Asn Thr Val Ser Thr Thr Thr Thr Leu Ala Pro Ser   1 5 10 15 Tyr Ser Leu Val Pro Gln Glu Thr Thr Ile Ser Tyr Ala Asp Asp Leu              20 25 30 Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg          35 40 45 <210> 32 <211> 119 <212> PRT <213> TFP11-Pro <400> 32 Leu Pro His Val Asp Val His Gln Glu Asp Ala His Gln His Lys Arg   1 5 10 15 Ala Val Ala Tyr Lys Tyr Val Tyr Glu Thr Val Val Val Asp Ser Asp              20 25 30 Gly His Thr Val Thr Pro Ala Ala Ser Glu Val Ala Thr Ala Ala Thr          35 40 45 Ser Ala Ile Ile Thr Thr Ser Val Leu Ala Pro Thr Ser Ser Ala Ala      50 55 60 Ala Ala Asp Ser Ser Ala Ser Ile Ala Val Ser Ser Ala Ala Leu Ala  65 70 75 80 Lys Asn Glu Lys Ile Ser Asp Ala Ala Ala Ser Ala Thr Ala Ser Thr                  85 90 95 Ser Gln Gly Ala Ser Ser Ser Ser Tyr Leu Ala Ala Ser Ala Ser Ala             100 105 110 Gly Leu Ala Leu Asp Lys Arg         115 <210> 33 <211> 177 <212> PRT <213> TFP12-Pro <400> 33 Met Ser Ser Asn Arg Leu Leu Lys Leu Ala Asn Lys Ser Pro Lys Lys   1 5 10 15 Ile Ile Pro Leu Lys Asp Ser Ser Phe Glu Asn Ile Leu Ala Pro Pro              20 25 30 His Glu Asn Ala Tyr Ile Val Ala Leu Phe Thr Ala Thr Ala Pro Glu          35 40 45 Ile Gly Cys Ser Leu Cys Leu Glu Leu Glu Ser Glu Tyr Asp Thr Ile      50 55 60 Val Ala Ser Trp Phe Asp Asp His Pro Asp Ala Lys Ser Ser Asn Ser  65 70 75 80 Asp Thr Ser Ile Phe Phe Thr Lys Val Asn Leu Glu Asp Pro Ser Lys                  85 90 95 Thr Ile Pro Lys Ala Phe Gln Phe Phe Gln Leu Asn Asn Val Pro Arg             100 105 110 Leu Phe Ile Phe Lys Leu Asn Ser Pro Ser Ile Leu Asp His Ser Val         115 120 125 Ile Ser Ile Ser Thr Asp Thr Gly Ser Glu Arg Met Lys Gln Ile Ile     130 135 140 Gln Ala Ile Lys Gln Phe Ser Gln Val Asn Asp Phe Ser Leu His Leu 145 150 155 160 Pro Val Gly Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys                 165 170 175 Arg     <210> 34 <211> 57 <212> PRT <213> TFP13-Pro <400> 34 Lys Lys Gly Glu His Asp Phe Thr Thr Thr Le Leu Thr Leu Ser Ser Asp   1 5 10 15 Gly Ser Leu Thr Thr Thr Thr Thr Ser Thr His Thr Thr His Lys Tyr Gly              20 25 30 Lys Phe Asn Lys Thr Ser Lys Ser Lys Thr Pro Leu Ala Ala Ser Ala          35 40 45 Ser Ala Gly Leu Ala Leu Asp Lys Arg      50 55 <210> 35 <211> 74 <212> PRT <213> TFP14-Pro <400> 35 His Asn Val Leu Leu Pro Ala Tyr Gly Arg Arg Cys Phe Phe Glu Asp   1 5 10 15 Leu Ser Lys Gly Asp Glu Leu Ser Ile Ser Phe Gln Phe Gly Asp Arg              20 25 30 Asn Pro Gln Ser Ser Ser Gln Leu Thr Gly Asp Phe Ile Ile Tyr Gly          35 40 45 Pro Glu Arg His Glu Val Leu Lys Thr Val Arg Glu Leu Ala Ala Ser      50 55 60 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg  65 70 <210> 36 <211> 156 <212> PRT <213> TFP16-Pro <400> 36 Ala Tyr Ala Pro Ser Glu Pro Trp Ser Thr Leu Thr Pro Thr Ala Thr   1 5 10 15 Tyr Ser Gly Gly Val Thr Asp Tyr Ala Ser Thr Phe Gly Ile Ala Val              20 25 30 Gln Pro Ile Ser Thr Thr Ser Ser Ala Ser Ser Ala Ala Thr Thr Ala          35 40 45 Ser Ser Lys Ala Lys Arg Ala Ala Ser Gln Ile Gly Asp Gly Gln Val      50 55 60 Gln Ala Ala Thr Thr Thr Ala Ser Val Ser Thr Lys Ser Thr Ala Ala  65 70 75 80 Ala Val Ser Gln Ile Gly Asp Gly Gln Ile Gln Ala Thr Thr Lys Thr                  85 90 95 Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Ile Gln Ala Thr             100 105 110 Thr Lys Thr Thr Ser Ala Lys Thr Thr Ala Ala Ala Val Ser Gln Ile         115 120 125 Ser Asp Gly Gln Ile Gln Ala Thr Thr Thr Thr Thr Leu Ala Pro Leu Ala     130 135 140 Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 145 150 155 <210> 37 <211> 190 <212> PRT <213> TFP17-Pro <400> 37 Ala Asn Ser Thr Thr Ser Ile Pro Ser Ser Cys Ser Ile Gly Thr Ser   1 5 10 15 Ala Thr Ala Thr Ala Gln Ala Asp Leu Asp Lys Ile Ser Gly Cys Ser              20 25 30 Thr Ile Val Gly Asn Leu Thr Ile Thr Gly Asp Leu Gly Ser Ala Ala          35 40 45 Leu Ala Ser Ile Gln Glu Ile Asp Gly Ser Leu Thr Ile Phe Asn Ser      50 55 60 Ser Ser Leu Ser Ser Phe Ser Ala Asp Ser Ile Lys Lys Ile Thr Gly  65 70 75 80 Asp Leu Asn Met Gln Glu Leu Ile Ile Leu Thr Ser Ala Ser Phe Gly                  85 90 95 Ser Leu Gln Glu Val Asp Ser Ile Asn Met Val Thr Leu Pro Ala Ile             100 105 110 Ser Thr Phe Ser Thr Asp Leu Gln Asn Ala Asn Asn Ile Val Ser         115 120 125 Asp Thr Thr Leu Glu Ser Val Glu Gly Phe Ser Thr Leu Lys Lys Val     130 135 140 Asn Val Phe Asn Ile Asn Asn Asn Arg Tyr Leu Asn Ser Phe Gln Ser 145 150 155 160 Ser Leu Glu Ser Val Ser Asp Ser Leu Gln Phe Ser Ser Asn Gly Asp                 165 170 175 Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg             180 185 190 <210> 38 <211> 74 <212> PRT <213> TFP19-Pro <400> 38 Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile Pro Ala   1 5 10 15 Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe Asp Val Ala              20 25 30 Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe Ile Asn          35 40 45 Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ala Ala Ser      50 55 60 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg  65 70 <210> 39 <211> 138 <212> PRT <213> TFP20-Pro <400> 39 Leu Ser Cys Glu Lys His Asp Val Leu Lys Lys Tyr Gln Val Gly Lys   1 5 10 15 Phe Ser Ser Leu Thr Ser Thr Glu Arg Asp Thr Pro Pro Ser Thr Thr              20 25 30 Ile Glu Lys Trp Trp Ile Asn Val Cys Glu Glu His Asn Val Glu Pro          35 40 45 Pro Glu Glu Cys Lys Lys Asn Asp Met Leu Cys Gly Leu Thr Asp Val      50 55 60 Ile Leu Pro Gly Lys Asp Ala Ile Thr Thr Gln Ile Ile Asp Phe Asp  65 70 75 80 Lys Asn Ile Gly Phe Asn Val Glu Glu Thr Glu Ser Ala Leu Thr Leu                  85 90 95 Thr Leu Asn Gly Ala Thr Trp Gly Ala Asn Ser Phe Asp Ala Lys Leu             100 105 110 Glu Phe Gln Cys Asn Asp Asn Met Lys Gln Asp Glu Leu Ala Ala Ser         115 120 125 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg     130 135 <210> 40 <211> 81 <212> PRT <213> TFP21-Pro <400> 40 Ala Pro Ala Val His His Ser Asp Asn His His His Asn Asp Lys Arg   1 5 10 15 Ala Val Val Thr Val Thr Gln Tyr Val Asn Ala Asp Gly Ala Val Val              20 25 30 Ile Pro Ala Ala Thr Thr Ala Thr Ser Ala Ala Ala Asp Gly Lys Val          35 40 45 Glu Ser Val Ala Ala Ala Thr Thr Thr Leu Ser Ser Thr Ala Ala Ala      50 55 60 Ala Thr Thr Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys  65 70 75 80 Arg     <210> 41 <211> 177 <212> PRT <213> TFP22-Pro <400> 41 Gln Phe Ser Asn Ser Thr Ser Ala Ser Ser Thr Asp Val Thr Ser Ser   1 5 10 15 Ser Ser Ile Ser Thr Ser Ser Gly Ser Val Thr Ile Thr Ser Ser Glu              20 25 30 Ala Pro Glu Ser Asp Asn Gly Thr Ser Thr Ala Ala Pro Thr Glu Thr          35 40 45 Ser Thr Glu Ala Pro Thr Thr Ala Ile Pro Thr Asn Gly Thr Ser Thr      50 55 60 Glu Ala Pro Thr Thr Ala Ile Pro Thr Asn Gly Thr Ser Thr Glu Ala  65 70 75 80 Pro Thr Asp Thr Thr Thr Glu Ala Pro Thr Thr Ala Leu Pro Thr Asn                  85 90 95 Gly Thr Ser Thr Glu Ala Pro Thr Asp Thr Thr Thr Glu Ala Pro Thr             100 105 110 Thr Gly Leu Pro Thr Asn Gly Thr Thr Ser Ala Phe Pro Pro Thr Thr         115 120 125 Ser Leu Pro Pro Ser Asn Thr Thr Thr Thr Pro Pro Tyr Asn Pro Ser     130 135 140 Thr Asp Tyr Thr Thr Asp Tyr Thr Val Val Thr Glu Tyr Thr Thr Tyr 145 150 155 160 Cys Pro Glu Arg Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys                 165 170 175 Arg     <210> 42 <211> 82 <212> PRT <213> TFP23-Pro <400> 42 Ile Gly Glu Leu Ala Phe Asn Leu Gly Val Lys Asn Asn Asp Gly Thr   1 5 10 15 Cys Lys Ser Thr Ser Asp Tyr Glu Thr Glu Leu Gln Ala Leu Lys Ser              20 25 30 Tyr Thr Ser Thr Val Lys Val Tyr Ala Ala Ser Asp Cys Asn Thr Leu          35 40 45 Gln Asn Leu Gly Pro Ala Ala Glu Ala Glu Gly Phe Thr Ile Phe Val      50 55 60 Gly Val Trp Pro Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp  65 70 75 80 Lys arg         <210> 43 <211> 47 <212> PRT <213> TFP24-Pro <400> 43 Ala Pro Ala Asn His Glu His Lys Asp Lys Arg Ala Val Val Thr Thr   1 5 10 15 Thr Val Gln Lys Gln Thr Thr Val Ile Val Asn Gly Ala Ala Ser Thr              20 25 30 Pro Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg          35 40 45 <210> 44 <211> 107 <212> PRT <213> TFP27-Pro <400> 44 Ala Ala Asn Val Thr Thr Ala Thr Val Ser Gln Glu Ser Thr Thr Leu   1 5 10 15 Val Thr Ile Thr Ser Cys Glu Asp His Val Cys Ser Glu Thr Val Ser              20 25 30 Pro Ala Leu Val Ser Thr Ala Thr Val Thr Val Asp Asp Val Ile Thr          35 40 45 Gln Tyr Thr Thr Trp Cys Pro Leu Thr Thr Glu Ala Pro Lys Asn Gly      50 55 60 Thr Ser Thr Ala Ala Pro Val Thr Ser Thr Glu Ala Pro Lys Asn Thr  65 70 75 80 Thr Ser Ala Ala Pro Thr His Ser Val Thr Ser Tyr Thr Leu Ala Ala                  85 90 95 Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg             100 105 <210> 45 <211> 62 <212> PRT <213> TFP29-Pro <400> 45 Glu Ser Ile Thr Thr Thr Ile Thr Ala Thr Lys Asn Gly His Val Tyr   1 5 10 15 Thr Lys Thr Val Thr Gln Asp Ala Thr Phe Val Trp Gly Gly Glu Asp              20 25 30 Ser Tyr Ala Ser Ser Thr Ser Ala Ala Glu Ser Ser Ala Ala Glu Thr          35 40 45 Ser Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg      50 55 60 <210> 46 <211> 119 <212> PRT <213> TFP37-Pro <400> 46 Ser Pro Ile Glu Asn Leu Phe Lys Tyr Arg Ala Val Lys Ala Ser His   1 5 10 15 Ser Lys Asn Ile Asn Ser Thr Leu Pro Ala Trp Asn Gly Ser Asn Ser              20 25 30 Ser Asn Val Thr Tyr Ala Asn Gly Thr Asn Ser Thr Thr Asn Thr Thr          35 40 45 Thr Ala Glu Ser Ser Gln Leu Gln Ile Ile Val Thr Gly Gly Gln Val      50 55 60 Pro Ile Thr Asn Ser Ser Leu Thr His Thr Asn Tyr Thr Arg Leu Phe  65 70 75 80 Asn Ser Ser Ser Ala Leu Asn Ile Thr Glu Leu Tyr Asn Val Ala Arg                  85 90 95 Val Val Asn Glu Thr Ile Gln Asp Asn Leu Ala Ala Ser Ala Ser Ala             100 105 110 Gly Leu Ala Leu Asp Lys Arg         115 <210> 47 <211> 93 <212> PRT <213> TFP38-Pro <400> 47 Thr Pro Pro Ala Cys Leu Leu Ala Cys Val Ala Gln Val Gly Lys Ser   1 5 10 15 Ser Ser Thr Cys Asp Ser Leu Asn Gln Val Thr Cys Tyr Cys Glu His              20 25 30 Glu Asn Ser Ala Val Lys Lys Cys Leu Asp Ser Ile Cys Pro Asn Asn          35 40 45 Asp Ala Asp Ala Ala Tyr Ser Ala Phe Lys Ser Ser Cys Ser Glu Gln      50 55 60 Asn Ala Ser Leu Gly Asp Ser Ser Gly Ser Ala Ser Ser Ser Val Leu  65 70 75 80 Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg                  85 90 <210> 48 <211> 113 <212> PRT <213> TFP39-Pro <400> 48 Ala Pro Ala Thr Thr Thr Leu Ser Pro Ser Asp Glu Arg Val Asn Leu   1 5 10 15 Val Glu Leu Gly Val Tyr Val Ser Asp Ile Arg Ala His Leu Ala Glu              20 25 30 Tyr Tyr Met Phe Gln Ala Ala His Pro Thr Glu Thr Tyr Pro Val Glu          35 40 45 Ile Ala Glu Ala Val Phe Asn Tyr Gly Asp Phe Thr Thr Met Leu Thr      50 55 60 Gly Ile Pro Ala Asp Gln Val Thr Arg Val Ile Thr Gly Val Pro Trp  65 70 75 80 Tyr Ser Thr Arg Leu Arg Pro Ala Ile Ser Ser Ala Leu Ser Lys Asp                  85 90 95 Gly Ile Tyr Thr Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys             100 105 110 Arg     <210> 49 <211> 72 <212> PRT <213> TFP41-Pro <400> 49 Thr Pro Leu Tyr Lys Arg Gln Asn Val Thr Ser Gly Gly Gly Thr Val   1 5 10 15 Pro Val Ile Ile Thr Gly Gly Pro Ala Val Ser Gly Ser Gln Ser Asn              20 25 30 Val Thr Thr Thr Thr Leu Phe Asn Ser Thr Ser Thr Leu Asn Ile Thr          35 40 45 Gln Leu Tyr Gln Ile Ala Thr Gln Val Asn Leu Ala Ala Ser Ala Ser      50 55 60 Ala Gly Leu Ala Leu Asp Lys Arg  65 70 <210> 50 <211> 100 <212> PRT <213> TFP42-Pro <400> 50 Gln Tyr Tyr Thr Asn Ser Ser Ser Ile Ala Ser Asn Ser Ser Thr Ala   1 5 10 15 Val Ser Ser Thr Ser Ser Gly Ser Val Ser Ile Ser Ser Ser Ile Glu              20 25 30 Leu Thr Ser Ser Thr Ser Asp Val Ser Ser Ser Leu Thr Glu Leu Thr          35 40 45 Ser Ser Ser Thr Glu Val Ser Ser Ser Ile Ala Pro Ser Thr Ser Ser      50 55 60 Ser Glu Val Ser Ser Ser Ile Thr Ser Ser Gly Ser Ser Val Ser Gly  65 70 75 80 Ser Ser Ser Ile Thr Ser Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala                  85 90 95 Leu Asp Lys Arg             100 <210> 51 <211> 69 <212> DNA <213> TFP1-Pre <400> 51 atgttcaatc gttttaacaa attccaagct gctgtcgctt tggccctact ctctcgcggc 60 gctctcggt 69 <210> 52 <211> 57 <212> DNA <213> TFP2-Pre <400> 52 atgacgccct atgcagtagc aattaccgtg gccttactaa ttgtaacagt gagcgca 57 <210> 53 <211> 63 <212> DNA <213> TFP3-Pre <400> 53 atgcaattca aaaacgtcgc cctagctgcc tccgttgctg ctctatccgc cactgcttct 60 gct 63 <210> 54 <211> 54 <212> DNA <213> TFP4-Pre <400> 54 atgagatttg cagaattctt ggtggtattt gccacgttag gcggggggat ggct 54 <210> 55 <211> 72 <212> DNA <213> TFP9-Pre <400> 55 atggtgttcg gtcagctgta tgcccttttc atcttcacgt tatcatgttg tatttccaaa 60 actgtgcaag ca 72 <210> 56 <211> 69 <212> DNA <213> TFP10-Pre <400> 56 atgttcaatc gttttaacaa attccaagct gctgtcgctt tggccctact ctctcgcggc 60 gctctcggt 69 <210> 57 <211> 57 <212> DNA <213> TFP11-Pre <400> 57 atgaaattct caactgccgt tactacgttg attagttctg gtgccatcgt gtctgct 57 <210> 58 <211> 66 <212> DNA <213> TFP12-Pre <400> 58 atgaattggc tgtttttggt ctcgctggtt ttcttctgcg gcgtgtcaac ccatcctgcc 60 ctggca 66 <210> 59 <211> 60 <212> DNA <213> TFP13-Pre <400> 59 atgaagttct cttctgttac tgctattact ctagccaccg ttgccaccgt tgccactgct 60                                                                           60 <210> 60 <211> 60 <212> DNA <213> TFP14-Pre <400> 60 atggcctcat ttgctactaa gtttgtcatt gcttgcttcc tgttcttctc ggcgtccgcc 60                                                                           60 <210> 61 <211> 54 <212> DNA <213> TFP16-Pre <400> 61 atgcaataca aaaagacttt ggttgcctct gctttggccg ctactacatt ggcc 54 <210> 62 <211> 57 <212> DNA <213> TFP17-Pre <400> 62 atgcaattca agaacgcttt gactgctact gctattctaa gtgcctccgc tctagct 57 <210> 63 <211> 57 <212> DNA <213> TFP19-Pre <400> 63 atgagatttc cttcaatttt tactgcagtt ttattcgcag catcctccgc attagct 57 <210> 64 <211> 57 <212> DNA <213> TFP20-Pre <400> 64 atggtatcga agacttggat atgtggcttc atcagtataa ttacagtggt acaggcc 57 <210> 65 <211> 51 <212> DNA <213> TFP21-Pre <400> 65 atgaaattat ccgctctatt agctttatca gcctccaccg ccgtcttggc c 51 <210> 66 <211> 54 <212> DNA <213> TFP22-Pre <400> 66 atgaaattat caactgtcct attatctgcc ggtttagcct cgactacttt ggcc 54 <210> 67 <211> 69 <212> DNA <213> TFP23-Pre <400> 67 atgcgtttct ctactacact cgctactgca gctactgcgc tatttttcac agcctcccaa 60 gtttcagct 69 <210> 68 <211> 57 <212> DNA <213> TFP24-Pre <400> 68 atgcgtctct ctaacctaat tgcttctgcc tctcttttat ctgctgctac tcttgct 57 <210> 69 <211> 54 <212> DNA <213> TFP27-Pre <400> 69 atgcaatttt ctactgtcgc ttctatcgcc gctgtcgccg ctgtcgcttc tgcc 54 <210> 70 <211> 54 <212> DNA <213> TFP29-Pre <400> 70 atgaaattct cttccgcttt ggttctatct gctgttgccg ctactgctct tgct 54 <210> 71 <211> 57 <212> DNA <213> TFP37-Pre <400> 71 atgaagttcc aagttgtttt atctgccctt ttggcatgtt catctgccgt cgtcgca 57 <210> 72 <211> 66 <212> DNA <213> TFP38-Pre <400> 72 atgcgtgcca tcactttatt atcttcagtc gtttctttgg cattgttgtc gaaggaagtc 60 ttagca 66 <210> 73 <211> 60 <212> DNA <213> TFP39-Pre <400> 73 atggtcaaac taacttcaat tgttgctggt gtcgctgcta ttgctgctgg tgtcgctgct 60                                                                           60 <210> 74 <211> 54 <212> DNA <213> TFP41-Pre <400> 74 atgaaattca catcagtgct agcatttttt cttgcaactt taacagcttc tgca 54 <210> 75 <211> 69 <212> DNA <213> TFP42-Pre <400> 75 atgttcaatc gctttaataa acttcaagcc gctttggctt tggtccttta ctcccaaagt 60 gcattgggc 69 <210> 76 <211> 285 <212> DNA <213> TFP1-Pro <400> 76 gactcttaca ccaatagcac ctcctccgca gacttgagtt ctatcacttc cgtctcgtca 60 gctagtgcaa gtgccaccgc ttccgactca ctttcttcca gtgacggtac cgtttatttg 120 ccatccacaa caattagcgg tgatctcaca gttactggta aagtaattgc aaccgaggcc 180 gtggaagtcg ctgccggtgg taagttgact ttacttgacg gtgaaaaata cgtcttctca 240 tctgaggccg cctcggcctc tgctggcctc gccttagata aaaga 285 <210> 77 <211> 333 <212> DNA <213> TFP2-Pro <400> 77 ctccaggtca acaattcatg tgtcgctttt ccgccatcaa atctcagggg caagaatgga 60 gacggtacta atgaacagta tgcaactgca ctactttcta ttccctggaa tgggcctcct 120 gagtcatcga gggatattaa tcttatcgaa ctcgaaccgc aagttgcact ctatttgctc 180 gaaaattata ttaaccatta ctacaacacc acaagagaca ataagtgccc taataaccac 240 tacctaatgg gagggcagtt gggtagctca tcggataata ggagtttgaa cgaggccgcc 300 tcggcctctg ctggcctcgc cttagataaa aga 333 <210> 78 <211> 288 <212> DNA <213> TFP3-Pro <400> 78 gaaggttaca ctccaggtga accatggtcc accttaaccc caaccggctc catctcttgt 60 ggtgcagccg aatacactac cacctttggt attgctgttc aagctattac ctcttcaaaa 120 gctaagagag acgttatctc tcaaattggt gacggtcaag tccaagccac ttctgctgct 180 actgctcaag ccaccgatag tcaagcccaa gctactacta ccgctacccc aaccagctcc 240 gaaaagatgg ccgcctcggc ctctgctggc ctcgccttag ataaaaga 288 <210> 79 <211> 132 <212> DNA <213> TFP4-Pro <400> 79 gcaccggttg agtctctggc cgggacccaa cggtatctgg tgcaaatgaa ggagcggttc 60 accacagaga agctgtgtgc tttggacgac aaggccgcct cggcctctgc tggcctcgcc 120 ttagataaaa ga 132 <210> 80 <211> 606 <212> DNA <213> TFP9-Pro <400> 80 gattcatcca aggaaagctc ttcctttatt tcgttcgaca aagagagtaa ctgggatacc 60 atcagcacta tatcttcaac ggcagatgtt atatcatccg ttgacagtgc tatcgctgtt 120 tttgaatttg acaatttctc attattggac agcttgatga ttgacgaaga atacccattc 180 ttcaatagat tctttgccaa tgatgtcagt ttaactgttc atgacgattc gcctttgaac 240 atctctcaat cattatctcc cattatggaa caatttactg tggatgaatt acctgaaagt 300 gcctctgact tactatatga atactcctta gatgataaaa gcatcgtttt gttcaagttt 360 acctcggatg cctacgattt gaaaaaatta gatgaattta ttgattcttg cttatcgttt 420 ttggaagata aatctggcga caatttgact gtggttatta actctcttgg ttgggctttt 480 gaagatgaag atggtgacga tgaatatgca acagaagaga ctttgagcca tcatgataac 540 aacaagggta aagaaggcga cgatctggcc gcctcggcct ctgctggcct cgccttagat 600 aaaaga 606 <210> 81 <211> 135 <212> DNA <213> TFP10-Pro <400> 81 atttattcaa acaatactgt ttctacaact accactttag cgcccagcta ctccttggtg 60 ccccaagaga ctaccatatc gtacgccgac gacctggccg cctcggcctc tgctggcctc 120 gccttagata aaaga 135 <210> 82 <211> 357 <212> DNA <213> TFP11-Pro <400> 82 ttaccacacg tggatgttca ccaagaagat gcccaccaac ataagagggc cgttgcgtac 60 aaatacgttt acgaaactgt tgttgtcgat tctgatggcc acactgtaac tcctgctgct 120 tcagaagtcg ctactgctgc tacctctgct atcattacaa catctgtgtt ggctccaacc 180 tcctccgcag ccgctgcgga tagctccgct tccattgctg tttcatctgc tgccttagcc 240 aagaatgaga aaatctctga tgccgctgca tctgccactg cctcaacatc tcaaggggca 300 tcctcctcat cctacctggc cgcctcggcc tctgctggcc tcgccttaga taaaaga 357 <210> 83 <211> 531 <212> DNA <213> TFP12-Pro <400> 83 atgtccagca acagactact aaagctggct aataaatctc ccaagaaaat tatacctctg 60 aaggactcaa gttttgaaaa catcttggca ccacctcacg aaaatgccta tatagttgct 120 ctgtttactg ccacagcgcc cgaaattggc tgttctctgt gtctcgagct agaatccgaa 180 tacgacacca tagtggcctc ctggtttgat gatcatccgg atgcaaaatc gtccaattcc 240 gatacatcta ttttcttcac aaaggtcaat ttggaggacc cttctaagac cattcctaaa 300 gcgttccagt ttttccaact aaacaatgtt cctagattgt tcatcttcaa actaaactct 360 ccctctattc tggaccacag cgtgatcagt atttccactg atactggctc agaaagaatg 420 aagcaaatca tacaagccat taagcagttc tcgcaagtaa acgacttctc tttacactta 480 cctgtgggtc tggccgcctc ggcctctgct ggcctcgcct tagataaaag a 531 <210> 84 <211> 171 <212> DNA <213> TFP13-Pro <400> 84 aagaagggtg aacatgattt cactaccact ttaactttgt catcggacgg tagtttaact 60 actaccacct ctactcatac cactcacaag tatggtaagt tcaacaagac ttccaagtcc 120 aagacccccc tggccgcctc ggcctctgct ggcctcgcct tagataaaag a 171 <210> 85 <211> 222 <212> DNA <213> TFP14-Pro <400> 85 cataatgtcc ttcttccagc ttatggccgt agatgcttct tcgaagactt gagtaagggt 60 gacgagctct ccatttcgtt ccagttcggt gatagaaacc ctcaatccag tagccagctg 120 actggtgact ttatcatcta cgggccggaa agacatgaag ttttgaaaac ggttagggaa 180 ctggccgcct cggcctctgc tggcctcgcc ttagataaaa ga 222 <210> 86 <211> 468 <212> DNA <213> TFP16-Pro <400> 86 gcctatgctc catctgagcc ttggtccact ttgactccaa cagccactta cagcggtggt 60 gttaccgact acgcttccac cttcggtatt gccgttcaac caatctccac tacatccagc 120 gcatcatctg cagccaccac agcctcatct aaggccaaga gagctgcttc ccaaattggt 180 gatggtcaag tccaagctgc taccactact gcttctgtct ctaccaagag taccgctgcc 240 gccgtttctc agatcggtga tggtcaaatc caagctacta ccaagactac cgctgctgct 300 gtctctcaaa ttggtgatgg tcaaattcaa gctaccacca agactacctc tgctaagact 360 accgccgctg ccgtttctca aatcagtgat ggtcaaatcc aagctaccac cactacttta 420 gcccctctgg ccgcctcggc ctctgctggc ctcgccttag ataaaaga 468 <210> 87 <211> 570 <212> DNA <213> TFP17-Pro <400> 87 gctaactcaa ctacttctat tccatcttca tgtagtattg gtacttctgc cactgctact 60 gctcaagctg atttggacaa aatctccggt tgtagtacca ttgttggtaa cttgaccatc 120 accggtgact tgggttccgc tgctttggct agtatccaag agattgatgg ttccttgact 180 atcttcaact ccagttcttt atcttctttc tccgctgact ctatcaagaa aatcaccggt 240 gatttgaaca tgcaagaatt gatcattttg accagtgctt ctttcggttc tttgcaagaa 300 gtagactcca ttaacatggt gactttgcct gccatttcta ccttctccac cgatttacaa 360 aatgctaaca acattattgt ttctgacacc actttggaaa gtgtcgaagg tttctccact 420 ttgaagaagg ttaatgtttt taacatcaac aacaacagat atctaaactc tttccaatct 480 tccttggaaa gtgtctctga ctctttacaa ttctcttcca acggtgacct ggccgcctcg 540 gcctctgctg gcctcgcctt agataaaaga 570 <210> 88 <211> 222 <212> DNA <213> TFP19-Pro <400> 88 gctccagtca acactacaac agaagatgaa acggcacaaa ttccggctga agctgtcatc 60 ggttacttag atttagaagg ggatttcgat gttgctgttt tgccattttc caacagcaca 120 aataacgggt tattgtttat aaatactact attgccagca ttgctgctaa agaagaaggg 180 gtggccgcct cggcctctgc tggcctcgcc ttagataaaa ga 222 <210> 89 <211> 414 <212> DNA <213> TFP20-Pro <400> 89 ttgtcctgcg agaagcatga tgtattgaaa aagtatcagg tgggaaaatt tagctcacta 60 acttctacgg aaagggatac tccgccaagc acaactattg aaaagtggtg gataaacgtt 120 tgcgaagagc ataacgtaga acctcctgaa gaatgtaaaa aaaatgacat gctatgtggt 180 ttaacagatg tcatcttgcc cggtaaggat gctatcacca ctcaaattat agattttgac 240 aaaaacattg gcttcaatgt cgaggaaact gagagtgcgc ttacattgac actaaacggc 300 gctacgtggg gcgccaattc ttttgacgca aaactagaat ttcagtgtaa tgacaatatg 360 aaacaagacg aactggccgc ctcggcctct gctggcctcg ccttagataa aaga 414 <210> 90 <211> 243 <212> DNA <213> TFP21-Pro <400> 90 gctccagctg tccaccatag tgacaaccac caccacaacg acaagcgtgc cgttgtcacc 60 gttactcagt acgtcaacgc agacggcgct gttgttattc cagctgccac caccgctacc 120 tcggcggctg ctgatggaaa ggtcgagtct gttgctgctg ccaccactac tttgtcctcg 180 actgccgccg ccgctacaac cctggccgcc tcggcctctg ctggcctcgc cttagataaa 240 aga 243 <210> 91 <211> 531 <212> DNA <213> TFP22-Pro <400> 91 caattttcca acagtacatc tgcttcttcc accgatgtca cttcctcctc ttccatctcc 60 acttcctctg gctcagtaac tatcacatct tctgaagctc cagaatccga caacggtacc 120 agcacagctg caccaactga aacctcaaca gaggctccaa ccactgctat cccaactaac 180 ggtacctcta ctgaagctcc aaccactgct atcccaacta acggtacctc tactgaagct 240 ccaactgata ctactactga agctccaacc accgctcttc caactaacgg tacttctact 300 gaagctccaa ctgatactac tactgaagct ccaaccaccg gtcttccaac caacggtacc 360 acttcagctt tcccaccaac tacatctttg ccaccaagca acactaccac cactcctcct 420 tacaacccat ctactgacta caccactgac tacactgtag tcactgaata tactacttac 480 tgtccggaac gggccgcctc ggcctctgct ggcctcgcct tagataaaag a 531 <210> 92 <211> 246 <212> DNA <213> TFP23-Pro <400> 92 attggtgaac tagcctttaa cttgggtgtc aagaacaacg atggtacttg taagtccact 60 tccgactatg aaaccgaatt acaagctttg aagagctaca cttccaccgt caaagtttac 120 gctgcctcag attgtaacac tttgcaaaac ttaggtcctg ctgctgaagc tgagggattt 180 actatctttg tcggtgtttg gccactggcc gcctcggcct ctgctggcct cgccttagat 240 aaaaga 246 <210> 93 <211> 141 <212> DNA <213> TFP24-Pro <400> 93 gcccccgcta accacgaaca caaggacaag cgtgctgtgg tcactaccac tgttcaaaaa 60 caaaccactg tcattgttaa tggtgccgct tcaactcccc tggccgcctc ggcctctgct 120 ggcctcgcct tagataaaag a 141 <210> 94 <211> 321 <212> DNA <213> TFP27-Pro <400> 94 gctgctaacg ttaccactgc tactgtcagc caagaatcta ccactttggt caccatcact 60 tcttgtgaag accacgtctg ttctgaaact gtctccccag ctttggtttc caccgctacc 120 gtcaccgtcg atgacgttat cactcaatac accacctggt gcccattgac cactgaagcc 180 ccaaagaacg gtacttctac tgctgctcca gttacctcta ctgaagctcc aaagaacacc 240 acctctgctg ctccaactca ctctgtcacc tcttacacgc tggccgcctc ggcctctgct 300 ggcctcgcct tagataaaag a 321 <210> 95 <211> 186 <212> DNA <213> TFP29-Pro <400> 95 gagagtatca ccaccaccat cactgccacc aagaacggtc atgtctacac taagactgtc 60 acccaagatg ctacttttgt ttggggtggt gaagactctt acgccagcag cacttctgcc 120 gctgaatctt ctgccgccga aacttcggcc gcctcggcct ctgctggcct cgccttagat 180 aaaaga 186 <210> 96 <211> 357 <212> DNA <213> TFP37-Pro <400> 96 agcccaatcg aaaacctatt caaatacagg gcagttaagg catctcacag taagaatatc 60 aactccactt tgccggcctg gaatgggtct aactctagca atgttaccta cgctaatgga 120 acaaacagta ctaccaatac tactactgcc gaaagcagtc aattacaaat cattgtaaca 180 ggtggtcaag taccaatcac caacagttct ttgacccaca caaactacac cagattattc 240 aacagttctt ctgctttgaa cattaccgaa ttgtacaatg ttgcccgtgt tgttaacgaa 300 acgatccaag ataacctggc cgcctcggcc tctgctggcc tcgccttaga taaaaga 357 <210> 97 <211> 279 <212> DNA <213> TFP38-Pro <400> 97 acacctccag cttgtttatt ggcctgtgtt gcgcaagtcg gcaaatcctc ttccacatgt 60 gactctttga atcaagtcac ctgttactgt gaacacgaaa actccgccgt caagaaatgt 120 ctagactcca tctgcccaaa caatgacgct gatgctgctt attctgcttt caagagttct 180 tgttccgaac aaaatgcttc attgggcgat tccagcggca gtgcctcctc atccgttctg 240 gccgcctcgg cctctgctgg cctcgcctta gataaaaga 279 <210> 98 <211> 339 <212> DNA <213> TFP39-Pro <400> 98 gccccagcca ccactacttt atctccctct gatgaaagag ttaacctggt cgaattaggt 60 gtctacgtct cagatatcag agctcatttg gctgaatact atatgttcca agctgctcat 120 ccaactgaaa cttacccagt tgaaattgct gaagctgttt tcaactacgg tgatttcacc 180 actatgttga ctggtattcc cgctgatcaa gtcactagag tcatcactgg tgtcccatgg 240 tactccacca gattgagacc agctatctcc agcgctctat ccaaggacgg tatctacacg 300 gccgcctcgg cctctgctgg cctcgcctta gataaaaga 339 <210> 99 <211> 216 <212> DNA <213> TFP41-Pro <400> 99 acaccacttt acaagaggca gaacgttact tctggcggcg gtacggtccc cgtgatcatc 60 acgggtggac ctgctgtatc tggtagccag tcaaacgtta ctaccacaac gctattcaac 120 tctacttcca ccttaaacat cactcaactt taccaaattg ctactcaagt taatctggcc 180 gcctcggcct ctgctggcct cgccttagat aaaaga 216 <210> 100 <211> 300 <212> DNA <213> TFP42-Pro <400> 100 caatattata ccaacagttc ctcaatcgct agtaacagct ccaccgccgt ttcgtcaact 60 tcatcaggtt ccgtttccat cagtagttct attgagttga cctcatctac ttctgatgtc 120 tcgagctctc tcactgagtt aacgtcatcc tccaccgaag tctcgagctc cattgctcca 180 tcaacctcgt cctctgaagt ctcgagctct attacttcat caggctcttc agtctccggc 240 tcatcttcta ttacttccct ggccgcctcg gcctctgctg gcctcgcctt agataaaaga 300                                                                          300 <210> 101 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer GAL100 <400> 101 gtatatggtg gtaatgccat g 21 <210> 102 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T1-H170 <400> 102 accgagagcg ccgcgagaga g 21 <210> 103 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T2-H171 <400> 103 tgcgctcact gttacaatta g 21 <210> 104 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T3-H172 <400> 104 agcagaagca gtggcggata g 21 <210> 105 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T4-H173 <400> 105 agccatcccc ccgcctaacg tg 22 <210> 106 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T9-H174 <400> 106 tgcttgcaca gttttggaaa tac 23 <210> 107 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T10-H175 <400> 107 cgctgacaca gaacttgcg 19 <210> 108 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T11-H176 <400> 108 agcagacacg atggcaccag 20 <210> 109 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T12-H177 <400> 109 tgccagggca ggatgggttg 20 <210> 110 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T13-H178 <400> 110 agcagtggca acggtggcaa c 21 <210> 111 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T14-H179 <400> 111 ggcggacgcc gagaagaac 19 <210> 112 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T16-H180 <400> 112 ggccaatgta gtagcggcc 19 <210> 113 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T17-H181 <400> 113 agctagagcg gaggcactta g 21 <210> 114 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T19-H182 <400> 114 agctaatgcg gaggatgctg cg 22 <210> 115 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T20-H183 <400> 115 ggcctgtacc actgtaatta t 21 <210> 116 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T21-H184 <400> 116 ggccaagacg gcggtggag 19 <210> 117 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T22-H185 <400> 117 ggccaaagta gtcgaggct 19 <210> 118 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T23-H186 <400> 118 agctgaaact tgggaggctg 20 <210> 119 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer T24-H187 <400> 119 agcaagagta gcagcagata aaag 24 <210> 120 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T27-H188 <400> 120 ggcagaagcg acagcggcg 19 <210> 121 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T29-H189 <400> 121 agcaagagca gtagcggca 19 <210> 122 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T37-H190 <400> 122 tgcgacgacg gcagatgaac 20 <210> 123 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T38-H191 <400> 123 tgctaagact tccttcgaca ac 22 <210> 124 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer T39-H192 <400> 124 agcagcgaca ccagcagca 19 <210> 125 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T41-H193 <400> 125 tgcagaagct gttaaagttg 20 <210> 126 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T42-H194 <400> 126 gcccaatgca ctttgggagt 20 <210> 127 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T1-H225 <400> 127 gactcttaca ccaatagcac 20 <210> 128 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T2-H226 <400> 128 ctccaggtca acaattcatg tg 22 <210> 129 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T3-H227 <400> 129 gaaggttaca ctccaggtga ac 22 <210> 130 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T4-H228 <400> 130 gcaccggttg agtctctggc 20 <210> 131 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T9-H229 <400> 131 gattcatcca aggaaagctc ttc 23 <210> 132 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T10-H230 <400> 132 atttattcaa acaatactgt ttc 23 <210> 133 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T11-H231 <133> 133 ttaccacacg tggatgttca c 21 <210> 134 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer T12-H232 <400> 134 atgtccagca acagactact aaag 24 <210> 135 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T13-H233 <400> 135 aagaagggtg aacatgattt cac 23 <210> 136 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T14-H234 <400> 136 cataatgtcc ttcttccagc 20 <210> 137 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T16-H235 <400> 137 gcctatgctc catctgagcc ttg 23 <210> 138 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T17-H236 <400> 138 gctaactcaa ctacttctat tc 22 <139> <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T19-H237 <400> 139 gctccagtca acactacaac 20 <210> 140 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T20-H238 <400> 140 ttgtcctgcg agaagcatga tg 22 <210> 141 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T21-H239 <400> 141 gctccagctg tccaccatag tg 22 <210> 142 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T22-H240 <400> 142 caattttcca acagtacatc tg 22 <210> 143 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T23-H241 <400> 143 attggtgaac tagcctttaa c 21 <210> 144 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T24-H242 <400> 144 gctcccgcta accacgaaca c 21 <210> 145 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T27-H243 <400> 145 gctgctaacg ttaccactgc tac 23 <210> 146 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T29-H244 <400> 146 gagagtatca ccaccaccat c 21 <210> 147 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T37-H245 <400> 147 agcccaatcg aaaacctatt c 21 <210> 148 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer T38-H246 <400> 148 acacctccag cttgtttatt gg 22 <210> 149 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T39-H247 <400> 149 gccccagcca ccactacttt atc 23 <210> 150 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T41-H248 <400> 150 acaccacttt acaagaggca g 21 <210> 151 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer T42-H249 <400> 151 caatattata ccaacagttc ctc 23 <210> 152 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer GT90R <400> 152 ggaaaggacc actcttacat aactag 26 <210> 153 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> primer GT70r <400> 153 tcagatttac agataatgat gtcattatta aatatatata tatatatatt gtcactccgt 60 tcaagtcgac 70 <210> 154 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer SH32 <400> 154 ctcgccttag ataaaagatt cccaaccatt cccttatc 38 <210> 155 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer SH33 <400> 155 cactccgttc aagtcgacct agaagccaca gctgccct 38 <210> 156 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer LNK39 <400> 156 ggccgcctcg gcctctgctg gcctcgcctt agataaaaga 40

Claims (12)

효모 유래의 단백질 분비융합인자의 프리-분비시그널 및 프로-분비시그널을 포함하는 외래단백질의 분비생산용 인공 단백질 분비융합인자로서, 상기 프리-분비시그널과 프로-분비시그널은 서로 다른 단백질 분비융합인자로부터 유래되는 것인 인공 단백질 분비융합인자.
Artificial protein secretion fusion factor for the production of foreign proteins including pre-secretion signal and pro-secretion signal of protein secretion fusion factor derived from yeast, wherein the pre-secretion signal and pro-secretion signal are different protein secretion fusion factor Artificial protein secretion fusion factor derived from.
제1항에 있어서,
상기 외래단백질은 인간 성장호르몬, 혈청단백질, 면역글로불린, 사이토카인, α-, β- 또는 γ-인터페론, 콜로니자극인자(GM-CSF), 혈소판 유도된 성장 인자(PDGF), 포스포리파제-활성화 단백질(PLAP), 인슐린, 종양 괴사 인자(TNF), 성장 인자, 호르몬, 칼시토닌(calcitonin), 칼시토닌 유전자 관련 펩타이드(Calcitonin Gene Related Peptide, CGPR), 엔케팔린(enkephalin), 소마토메딘, 에리스로포이에틴, 시상하부 분비 인자, 프롤락틴, 만성 고나도트로핀, 조직 플라스미노겐 활성화제, 성장호르몬 분비 펩타이드(growth hormone releasing peptide; GHPR), 흉선 체액성 인자(thymic humoral factor; THF), 아스파라기나제, 아르기나제, 아르기닌 데아미나제, 아데노신 데아미나제, 과산화물 디스뮤타제, 엔도톡시나제, 카탈라제, 키모트립신, 리파제, 우리카제, 아데노신 디포스파타제, 티로시나제, 빌리루빈 옥시다제, 글루코즈 옥시다제, 글루코다제, 갈락토시다제, 글루코세레브로시다제 및 글루코우로니다제로 구성된 군으로부터 선택되는 것인 인공 단백질 분비융합인자.
The method of claim 1,
The foreign protein may be human growth hormone, serum protein, immunoglobulin, cytokine, α-, β- or γ-interferon, colony stimulating factor (GM-CSF), platelet induced growth factor (PDGF), phospholipase-activation Protein (PLAP), insulin, tumor necrosis factor (TNF), growth factor, hormone, calcitonin, calcitonin gene related peptide (CGPR), enkephalin, somatomedin, erythropoietin, hypothalamus Secretion factor, prolactin, chronic gonadotropin, tissue plasminogen activator, growth hormone releasing peptide (GHPR), thymic humoral factor (THF), asparaginase, arginase Arginine deaminase, adenosine deaminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bili Blank oxidase, glucose oxidase, glucose oxidase, galactosidase, glucosidase to let the celebrity glucoside and right is the artificial fusion protein secretion factor is selected from the group consisting of zero.
제1항에 있어서,
상기 프리-분비시그널은 서열번호 1 내지 25로 구성된 군으로부터 선택되는 단백질 또는 서열번호 51 내지 75로 구성된 군으로부터 선택되는 유전자인 것인 인공 단백질 분비융합인자.
The method of claim 1,
The pre-secretion signal is an artificial protein secretion fusion factor is a protein selected from the group consisting of SEQ ID NO: 1 to 25 or a gene selected from the group consisting of SEQ ID NO: 51 to 75.
제1항에 있어서,
상기 프로-분비시그널은 서열번호 26 내지 50으로 구성된 군으로부터 선택되는 단백질 또는 서열번호 76 내지 100으로 구성된 군으로부터 선택되는 유전자인 것인 인공 단백질 분비융합인자.
The method of claim 1,
The pro-secretion signal is an artificial protein secretion fusion factor that is a protein selected from the group consisting of SEQ ID NO: 26 to 50 or selected from the group consisting of SEQ ID NO: 76 to 100.
제1항에 있어서,
상기 외래단백질은 인간 성장호르몬이고, 상기 프리-분비시그널은 TFP20-Pre(서열번호 14 및 64), TFP3-Pre(서열번호 3 및 53) 또는 TFP14-Pre(서열번호 10 및 60)이며, 상기 프로-분비시그널은 TFP19-Pro(서열번호 38 및 88) 또는 TFP24-Pro(서열번호 43 및 93)인 것인 인공 단백질 분비융합인자.
The method of claim 1,
The foreign protein is human growth hormone, and the pre-secretion signal is TFP20-Pre (SEQ ID NOs: 14 and 64), TFP3-Pre (SEQ ID NOs: 3 and 53) or TFP14-Pre (SEQ ID NOs: 10 and 60). The pro-secretory signal is an artificial protein secretion fusion factor of TFP19-Pro (SEQ ID NOs: 38 and 88) or TFP24-Pro (SEQ ID NOs: 43 and 93).
i) 제1항 내지 제5항 중 어느 한 항에 따른 인공 단백질 분비융합인자 및 생산하고자 하는 외래 단백질 유전자를 포함하는 발현벡터를 제조하는 단계;
ii) 상기 i) 단계의 발현벡터를 효모에 도입하여 형질전환체를 수득하는 단계; 및
iii) 상기 형질전환체를 배양하고, 이의 배양물 또는 배양상등액으로부터 외래단백질을 회수하는 단계를 포함하는, 외래 단백질을 생산하는 방법.
i) preparing an expression vector comprising the artificial protein secretion fusion factor according to any one of claims 1 to 5 and the foreign protein gene to be produced;
ii) introducing the expression vector of step i) into yeast to obtain a transformant; And
iii) culturing the transformant and recovering the foreign protein from its culture or culture supernatant.
제6항에 있어서,
상기 발현벡터는 도 6의 개열지도로 표시되는 플라스미드 YGaC9-hGH인 것인 방법.
The method according to claim 6,
The expression vector is plasmid YGaC9-hGH represented by the cleavage map of FIG.
제6항에 있어서,
상기 형질전환체의 배양은 비-이온성 계면활성제를 포함하는 배지를 이용하여 수행되는 것인 방법.
The method according to claim 6,
Culturing the transformant is carried out using a medium comprising a non-ionic surfactant.
제8항에 있어서,
상기 비-이온성 계면활성제는 폴리소르베이트(Polysorbate) 또는 폴록사머(poloxamer)인 것인 방법.
9. The method of claim 8,
Wherein said non-ionic surfactant is polysorbate or poloxamer.
제6항에 있어서,
회수한 외래단백질의 정제 단계를 추가로 포함하는 것인 방법.
The method according to claim 6,
The method further comprises the step of purifying the recovered foreign protein.
제10항에 있어서,
상기 외래단백질은 인간 성장호르몬이며, 상기 정제는 음이온 교환 크로마토 그래피, 소수성 상호작용 크로마토그래피, 음이온 교환 크로마토그래피 및 젤여과 크로마토그래피의 순서로 정제하는 것인 방법.
The method of claim 10,
The foreign protein is human growth hormone, and the purification is purified in the order of anion exchange chromatography, hydrophobic interaction chromatography, anion exchange chromatography and gel filtration chromatography.
(ⅰ) 제1항 내지 제5항 중 어느 한 항에 따른 인공 단백질 분비융합인자를 포함하는 라이브러리에 목적하는 외래단백질의 유전자를 도입하여 각각의 발현벡터를 수득하는 단계;
(ⅱ) 상기 각 발현벡터를 효모에 도입하여 각각의 형질전환체를 수득하는 단계;
(ⅲ) 수득한 각 형질전환체를 배양하여 외래단백질을 분비생산하는 단계;
(ⅳ) 분비생산된 외래단백질의 양에 따라 외래단백질의 분비생산 효율이 우수한 형질전환체를 선별하는 단계; 및
(ⅴ) 상기 선별된 형질전환체에 포함된 발현벡터를 분석하여 프리-분비시그널 및 프로-분비시그널의 조합을 결정하는 단계를 포함하는, 외래단백질의 분비생산에 적합한 인공 단백질 분비융합인자의 스크리닝 방법.(Iii) introducing a gene of a foreign protein of interest into a library comprising an artificial protein secretion fusion factor according to any one of claims 1 to 5 to obtain respective expression vectors;
(Ii) introducing each of said expression vectors into yeast to obtain respective transformants;
(Iii) culturing the obtained transformants to secrete and produce foreign proteins;
(Iii) selecting a transformant having excellent secretion efficiency of the foreign protein according to the amount of the secreted foreign protein produced; And
(Iii) screening an artificial protein secretion fusion factor suitable for the production of foreign proteins, comprising determining a combination of pre-secretion signal and pro-secretion signal by analyzing the expression vector included in the selected transformant. Way.
KR1020110060407A 2011-06-21 2011-06-21 A novel fabricated translational fusion partner for secretory production of foreign protein, a process for preparing the foreign protein using the same and a screening method of the same KR101653809B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020110060407A KR101653809B1 (en) 2011-06-21 2011-06-21 A novel fabricated translational fusion partner for secretory production of foreign protein, a process for preparing the foreign protein using the same and a screening method of the same

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020110060407A KR101653809B1 (en) 2011-06-21 2011-06-21 A novel fabricated translational fusion partner for secretory production of foreign protein, a process for preparing the foreign protein using the same and a screening method of the same

Publications (2)

Publication Number Publication Date
KR20120140577A true KR20120140577A (en) 2012-12-31
KR101653809B1 KR101653809B1 (en) 2016-09-05

Family

ID=47906600

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020110060407A KR101653809B1 (en) 2011-06-21 2011-06-21 A novel fabricated translational fusion partner for secretory production of foreign protein, a process for preparing the foreign protein using the same and a screening method of the same

Country Status (1)

Country Link
KR (1) KR101653809B1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016017736A1 (en) * 2014-07-31 2016-02-04 国立大学法人神戸大学 Secretion signal peptide, and protein secretory production and cell surface display using said secretion signal peptide
CN115197922A (en) * 2021-04-09 2022-10-18 派格生物医药(苏州)股份有限公司 Uricase or its isodynamic and its preparing method
CN115197922B (en) * 2021-04-09 2024-05-14 派格生物医药(苏州)股份有限公司 Uricase or its isoforms and methods of making same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GenBank Accession Number AGW24992 (2013.09.25.) *
손정훈 등. 과제보고서, 특정기초연구사업. 한국과학재단. 페이지 1-86 (2007.11.09)* *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016017736A1 (en) * 2014-07-31 2016-02-04 国立大学法人神戸大学 Secretion signal peptide, and protein secretory production and cell surface display using said secretion signal peptide
JPWO2016017736A1 (en) * 2014-07-31 2017-05-18 国立大学法人神戸大学 Secretion signal peptide and protein secretion and cell surface display using it
CN115197922A (en) * 2021-04-09 2022-10-18 派格生物医药(苏州)股份有限公司 Uricase or its isodynamic and its preparing method
CN115197922B (en) * 2021-04-09 2024-05-14 派格生物医药(苏州)股份有限公司 Uricase or its isoforms and methods of making same

Also Published As

Publication number Publication date
KR101653809B1 (en) 2016-09-05

Similar Documents

Publication Publication Date Title
CN102239183B (en) The screening of the albumen of a large amount of secretions is applied as fusion partner with them in recombinant protein preparation
KR100975596B1 (en) Library of translational fusion partners for producing recombinant proteins and translational fusion partners screened therefrom
AU2013341049B2 (en) Expression sequences
JP3730256B2 (en) Secretion signal gene and expression vector having the same
EP0123544A2 (en) Process for expressing heterologous protein in yeast, expression vehicles and yeast organisms therefor
JPH02163074A (en) Recombinant saccharomyces
US9012367B2 (en) Rapid screening method of translational fusion partners for producing recombinant proteins and translational fusion partners screened therefrom
AU615451B2 (en) Immunogenic recombinant yeast expression product and method for purifying it
WO2013111754A1 (en) Expression vector and method for producing protein
Razis et al. The periplasmic expression of recombinant human epidermal growth factor (hEGF) in Escherichia coli
KR101653809B1 (en) A novel fabricated translational fusion partner for secretory production of foreign protein, a process for preparing the foreign protein using the same and a screening method of the same
KR101802980B1 (en) Novel translational fusion partner derived from Pichia pastoris for secretory production of foreign proteins and use thereof
Müller II et al. Production of IFNα-2a in Hansenula polymorpha
KR100915670B1 (en) A novel YlMPO1 gene derived from Yarrowia lipolytica and a process for preparing a glycoprotein not being mannosylphosphorylated by using a mutated Yarrowia lipolytica in which YlMPO1 gene is disrupted
KR100618563B1 (en) Method for Producing Recombinant Proteins in Yeast using cellulose-binding domain
KR100798894B1 (en) Translational fusion partner for producing recombinant proteins
WO2020182768A1 (en) Methods for bioactive lectin production with improved yield
WO2011051964A2 (en) Process for recombinant human growth hormone
US20230097374A1 (en) Recombinant expression platform, constructs and methods for expression of Difficult to Express Proteins (DTE-Ps)
KR0171161B1 (en) Expression vector and process for the preparation of human calcitonin precursors
KR20020066482A (en) Secretion system of recombinant protein containing sequence enhancer and the promoter of methylotropic yeast
EP0188555A1 (en) Yeast cloning vehicle
CN114957398A (en) Preparation method of surface protein receptor binding region of alpha strain 2019-nCoV
KR20050075661A (en) Rapid screening method of suitable translational fusion partners for producing recombinant proteins
KR20050076139A (en) Translational fusion partners for the secretory production of proteins

Legal Events

Date Code Title Description
A201 Request for examination
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right
GRNT Written decision to grant