公开(公告)号：KR100381381B1

公开(公告)日：2003-12-31

申请号：KR1019980051563

申请日：1998-11-28

Applicant: 한국과학기술원

Inventor： 이상엽 ,  최종현 ,  정기준 ,  김선창

IPC: C12N15/74 ,  C12N1/21

Abstract: PURPOSE: Recombinant plasmids having secretion signal sequence of endoxylanase from Bacillus are provided which express and secret foreign protein in transformed E. coli therewith. CONSTITUTION: A secretion signal sequence of endoxylanase fragment from plasmid, pKJX4, containing endoxylase gene from Bacillus is amplified by PCR. Recombinant plasmid, pJS101, is constructed by cloning PCR fragments into plasmid, pTrc99A, having strong trc promoter. Plasmid pJS101 contains signal sequence of endoxylanase and endoxylanase joined with PstI site. 3' end of endoxylanase has restriction sites of BamHI, XbaI, SalI, PstI and HindIII for the cloning of foreign protein. Plasmid pJS101DeltaP is constructed by removing PstI site of 3' end of endoxylanase gene for the easy introduction of foreign protein. T7 promoter containing vector, pJS301, is constructed by joining of NdeI and BamHI digested fragment from pJS101DeltaP and pET21c. Induction experiment with IPTG shows that the yield of secreted protein is at least 6% of total protein. Alkaline phosphatase is over expressed and constitutes 6.9% of total protein.

Abstract translation: 目的：提供具有来自芽孢杆菌属的木聚糖内切酶分泌信号序列的重组质粒，其在转化的大肠杆菌中表达并分泌外源蛋白质。 构成：通过PCR扩增来自质粒pKJX4的含有来自杆菌的内切酶基因的木聚糖内切酶片段的分泌信号序列。 通过将PCR片段克隆到具有强trc启动子的质粒pTrc99A中构建重组质粒pJS101。 质粒pJS101含有内切木聚糖酶的信号序列和与PstI位点连接的木聚糖内切酶。 内切木聚糖酶的3'末端具有BamHI，XbaI，SalI，PstI和HindIII的限制位点用于克隆外源蛋白。 质粒pJS101DeltaP是通过去除内切木聚糖酶基因的3'末端的PstI位点以便于引入外来蛋白质而构建的。 含有T7启动子的载体pJS301通过连接来自pJS101DeltaP和pET21c的NdeI和BamHI消化的片段构建。 用IPTG诱导实验显示分泌蛋白的产量至少为总蛋白的6％。 碱性磷酸酶过度表达并占总蛋白的6.9％。

公开(公告)号：KR1020030014959A

公开(公告)日：2003-02-20

申请号：KR1020010048881

申请日：2001-08-14

Applicant: 한국과학기술원

Inventor： 이상엽 ,  정기준

IPC: C12N15/70

CPC classification number: C12P21/02 ,  C07K14/245 ,  C07K14/675 ,  C07K2319/02 ,  C07K2319/035 ,  C07K2319/75 ,  C12N15/625 ,  C12N15/70

Abstract: PURPOSE: A process for producing a target protein from Escherichia coli through the extracellular secretion using an OmpF gene is provided, therefor the target protein can be simply and massively produced from Escherichia coli through the high concentration of cell cultivation. CONSTITUTION: The process for producing a target protein from Escherichia coli through the extracellular secretion using the OmpF gene comprises the steps of: (i) cloning an oligopeptide expressing a protease-recognition and digestion site and a gene encoding a target protein into an expression vector pOmpF6 containing an ampicillin-resistant gene, an OmpF promoter and the OmpF gene to construct a recombinant expression vector; (ii) transforming a host cell without the OmpF gene with the recombinant expression vector to prepare a transformed cell; (iii) culturing the transformed cell to obtain a OmpF-fusion protein from the cultivated medium; and (iv) treating the OmpF-fusion protein with protease to recover the target protein, wherein the protease is factor Xa, enterokinase, genase, IgA protease, intein, thrombin, trypsin, pepsin, subtilisin or plasmin.

Abstract translation: 目的：提供通过使用OmpF基因通过细胞外分泌从大肠杆菌产生靶蛋白的方法，由此可以通过高浓度的细胞培养从大肠杆菌中简单大量地产生靶蛋白。 构成：使用OmpF基因通过细胞外分泌从大肠杆菌产生靶蛋白的方法包括以下步骤：（i）将表达蛋白酶识别和消化位点的寡肽和编码靶蛋白的基因克隆到表达载体中 含有氨苄青霉素抗性基因的pOmpF6，OmpF启动子和OmpF基因构建重组表达载体; （ii）用重组表达载体转化没有OmpF基因的宿主细胞以制备转化细胞; （iii）培养转化细胞以从培养基中获得OmpF融合蛋白; 蛋白酶是因子Xa，肠激酶，基因酶，IgA蛋白酶，内含肽，凝血酶，胰蛋白酶，胃蛋白酶，枯草杆菌蛋白酶或纤溶酶，并且（iv）用蛋白酶处理OmpF-融合蛋白以回收靶蛋白。

公开(公告)号：KR1020010094652A

公开(公告)日：2001-11-01

申请号：KR1020000017052

申请日：2000-03-31

Applicant: 한국과학기술원

Inventor： 이상엽 ,  정기준

IPC: C12N15/70

CPC classification number: C07K14/535 ,  C07K2319/00 ,  C12N15/70

Abstract: PURPOSE: An E.coli secreting human granulocyte-colony stimulating factor(hG-CSF) is provided, thereby secreting the hG-CSF effectively into cytoplasm and easily producing it through the simple processes. CONSTITUTION: A plasmid vector pTHKCSFmII comprises Kanamycin-tolerance gene, endozylanase secreting signal sequence, nucleotide sequence of SEQ ID NO: 1 encoding oligopeptide, human granulocyte-colony stimulating factor(hG-CSF) gene comprising the nucleotide sequence of SEQ ID NO: 26 encoding N-terminal, and Trc promoter, wherein the C-terminal of oligopeptide is Ile-Glu-Gly-Arg. E.coli MC4100/pTHKCSFmII (KCTC 0754BP) is transformed with plasmid vector pTHKCSFmII, wherein E.coli is selected from the group consisting of E.coli XL1-Blue, E.coli MC4100, E.coliBL21(DE3) , E.coli HB101 and E.coli W3110. The method for producing hG-CSF comprises the steps of: incubating E.coli MC4100/pTHKCSFmII (KCTC 0754BP) to produce hG-CSF fusion proteins; and treating hG-CSF with protease to produce hG-CSF.

Abstract translation: 目的：提供分泌人粒细胞集落刺激因子（hG-CSF）的大肠杆菌，从而通过简单的过程将hG-CSF有效分泌到细胞质中并容易地产生。 构成：质粒载体pTHKCSFmII包含卡那霉素耐受基因，内切酶分泌信号序列，编码寡肽的SEQ ID NO：1的核苷酸序列，包含SEQ ID NO：26的核苷酸序列的人粒细胞集落刺激因子（hG-CSF）基因 编码N末端和Trc启动子，其中寡肽的C-末端是Ile-Glu-Gly-Arg。 大肠杆菌MC4100 / pTHKCSFmII（KCTC 0754BP）用质粒载体pTHKCSFmII转化，其中大肠杆菌选自大肠杆菌XL1-Blue，大肠杆菌MC4100，大肠杆菌BL21（DE3），大肠杆菌 HB101和大肠杆菌W3110。 生产hG-CSF的方法包括以下步骤：培养大肠杆菌MC4100 / pTHKCSFmII（KCTC 0754BP）以产生hG-CSF融合蛋白; 并用蛋白酶处理hG-CSF以产生hG-CSF。