公开(公告)号：KR100253913B1

公开(公告)日：2000-04-15

申请号：KR1019970073899

申请日：1997-12-26

Applicant: 한국과학기술원

Inventor： 김학성 ,  김근중 ,  이동철

IPC: C12N15/52

Abstract: PURPOSE: Provided is a gene of D-hydantoinase from Bacillus stearothermophilus SD-1 (KCTC 8551P) to manufacture proteins in a high yield. The D-hydantoinase is very useful in manufacturing D-amino acid used in drugs and agricultural chemicals etc. CONSTITUTION: A gene of D-hydantoinase is obtained by the following steps of: i) separating a gene that codes D-hydantoinase from Bacillus stearothermophilus SD-1 KCTC 8551P and cloning the gene with plasmid pBR32; ii) inserting a 3.1kb gene segment that codes D-hydantoinase into plasmid pUC18 to make recombinant plasmid pHBU183 and transforming E. coli with the plasmid to obtain fused protein; iii) to analyze the D-hydantoinase coding gene, cutting 3.1kb gene segment inserted into plasmid pHBU183 with restriction enzymes EcoRI and SacI, and linking new segments with plasmid pBluescript SK to make plasmid pHBMT183; iv) removing unnecessary DNA segments, cutting plasmid pHBMT183 with restriction enzyme EcoRI, and recovering DNA segments with Exonuclease III; v) linking recovered segments with pBluescript SK and inserting the plasmid into E. coli XL1-Blue for base analysis; vi) using the plasmid as a template to determine base sequences with primers T3 and T7.

Abstract translation: 目的：提供嗜热脂肪芽孢杆菌SD-1（KCTC 8551P）的D-乙内酰脲酶基因，高产量制备蛋白质。 D-乙内酰脲酶在制造用于药物和农药等的D-氨基酸中非常有用。构成：通过以下步骤获得D-乙内酰脲酶的基因：i）从嗜热脂肪芽孢杆菌中分离出编码D-乙内酰脲酶的基因 SD-1 KCTC 8551P，用质粒pBR32克隆该基因; ii）将编码D-乙内酰脲酶的3.1kb基因片段插入质粒pUC18以制备重组质粒pHBU183并用质粒转化大肠杆菌以获得融合蛋白; iii）分析D-乙内酰脲酶编码基因，用限制酶EcoRI和SacI切割插入质粒pHBU183的3.1kb基因片段，并将新片段与质粒pBluescript SK连接，制备质粒pHBMT183; iv）去除不必要的DNA片段，用限制酶EcoRI切割质粒pHBMT183，并用外切核酸酶III回收DNA片段; v）将回收的片段与pBluescript SK连接并将质粒插入大肠杆菌XL1-Blue进行碱基分析; vi）以质粒为模板，用引物T3和T7测定碱基序列。

公开(公告)号：KR100428998B1

公开(公告)日：2004-04-28

申请号：KR1020010055394

申请日：2001-09-10

Applicant: 한국과학기술원

Inventor： 김학성 ,  김근중 ,  천영훈 ,  이동은

IPC: C07K1/00

CPC classification number: C12N15/1058 ,  C07K14/43595 ,  C12N9/86 ,  C12N15/102

Abstract: The present invention relates to a method for manufacturing a mutant library of proteins with various sizes and sequences from a parental protein, microorganisms transformed with plasmids containing recombinant DNAs prepared by the insertion of a genomic DNA fragment into a defective template, a process for preparing proteins with different sizes and sequences from the parental protein which comprises the steps of culturing the transformed microorganisms and obtaining desired proteins from the culture, and proteins prepared by the said process. In accordance with the invention, a mutant library of proteins with various sizes and sequences can be manufactured from a parental protein in an efficient and simple manner, by constructing a library of microorganisms transformed with recombinant plasmids containing E. coli genomic DNA fragments inserted into defective genes and selecting clones expressing proteins with restored functions or modified characteristics.

Abstract translation: 本发明涉及用于从亲代蛋白质​​制备具有各种大小和序列的蛋白质突变体文库的方法，用包含通过将基因组DNA片段插入缺陷模板中制备的重组DNA的质粒转化的微生物，制备蛋白质的方法 具有来自亲本蛋白质的不同大小和序列，其包括培养转化的微生物并从培养物获得期望的蛋白质的步骤，以及通过所述方法制备的蛋白质。 根据本发明，通过构建用含有插入缺陷中的大肠杆菌基因组DNA片段的重组质粒转化的微生物文库，可以以有效且简单的方式从亲本蛋白质制备具有各种大小和序列的蛋白质突变体文库 基因并选择表达具有恢复功能或修饰特征的蛋白质的克隆。

公开(公告)号：KR1020030022447A

公开(公告)日：2003-03-17

申请号：KR1020010055394

申请日：2001-09-10

Applicant: 한국과학기술원

Inventor： 김학성 ,  김근중 ,  천영훈 ,  이동은

IPC: C07K1/00

CPC classification number: C12N15/1058 ,  C07K14/43595 ,  C12N9/86 ,  C12N15/102

Abstract: PURPOSE: A method for manufacturing mutant library of proteins with different sequence and size from a mother protein is provided, thereby the mutant library of proteins can be useful for biotechnology. CONSTITUTION: The method for manufacturing mutant library of proteins with different sequence and size from the mother protein comprises the steps of: (i) inducing mutation to a gene encoding the mother protein to construct a defective template of which function is lost; (ii) inserting a randomly synthesized oligonucleotide sequence or a restriction enzyme-treated genomic DNA into the defective template to construct a recombinant plasmid, and transforming microorganism with the recombinant plasmid to prepare a transformed microorganism library; and (iii) selecting salvage protein of which distinct function is recovered or characteristic is changed from the proteins expressed from the transformed microorganism library, wherein the mutation is induced by deletion or frame shift of nucleotides.

Abstract translation: 目的：提供从母体蛋白质制备具有不同序列和大小的蛋白质突变文库的方法，因此蛋白质突变文库可用于生物技术。 构成：从母体蛋白质制备具有不同序列和大小的蛋白质的突变型文库的方法包括以下步骤：（i）诱导编码母体蛋白质的基因的突变以构建失去功能的缺陷模板; （ii）将随机合成的寡核苷酸序列或限制酶处理的基因组DNA插入缺陷模板中以构建重组质粒，并用重组质粒转化微生物以制备转化的微生物文库; 和（iii）从转化的微生物文库表达的蛋白质中选择回收不同功能或特征的补救蛋白，其中通过核苷酸的缺失或帧移位诱导突变。

公开(公告)号：KR1019990054117A

公开(公告)日：1999-07-15

申请号：KR1019970073899

申请日：1997-12-26

Applicant: 한국과학기술원

Inventor： 김학성 ,  김근중 ,  이동철

IPC: C12N15/52

Abstract: 본 발명은 고온성 세균인 바실러스 스티아로더모필러스 SD-1(Bacillus stearothermophilus SD-1, KCTC 8551P)에서 유래한 내열성 D-히단토이나아제(D-hydantoinase)를 암호화하는 신규한 염기서열 및 그로부터 번역되는 아미노산 서열에 관한 것이다. 본 발명의 내열성 D-히단토이나아제는 공지의 상온균 및 고온균 유래의 어떤 효소와도 그 염기서열이나 아미노산 서열에 있어서 상이하며 특히, 카르복실기 말단의 아미노산 서열 TSTISKQSEELTI을 특징적으로 갖고 있는 바, 이를 유전공학기술에 의해 대량 발현시켜, 의약품, 농약, 생리활성물질 등의 합성에 이용되는 D-아미노산의 제조에 효율적으로 사용한다.