公开(公告)号：KR1020010019100A

公开(公告)日：2001-03-15

申请号：KR1019990035354

申请日：1999-08-25

Applicant: 동화약품주식회사

Inventor： 임경묵 ,  차선신 ,  오병하 ,  윤제인 ,  이경재 ,  김종우 ,  윤성준

IPC: C12N15/12

CPC classification number: C12N15/70 ,  C07K14/47

Abstract: PURPOSE: A method for producing TNF Related Apoptosis Inducing Ligand(TRAIL) protein, and for crystallizing the TRAIL protein and the three-dimensional structure thereof are provided, thereby TRAIL protein having improved activity, which specifically kills cancer cells and cells infected by virus, can be produced. CONSTITUTION: The TRAIL having sequence No. 2 is produced by the steps of: inserting the human TRAIL protein gene into a plasmid to produce pET3a-TLS; transforming E. coli with pET3a-TLS to produce E. coli BL21-TLS(pET3a-TLS)(KCCM-10169); incubating the transformed E. coli BL21-TLS(pET3a-TLS)(KCCM-10169) at 22 to 37 deg. C and adding IPTG during incubation to express the TRAIL gene; breaking the cells, collecting insoluble fragments, denaturing and refolding the fragments to collect soluble protein; treating the soluble fragment and soluble protein with cationic exchange chromatography and size excluding chromatography to isolate and purify the TRAIL protein. The TRAIL protein is crystallized by using a solution containing 20 to 40% of PEG MME550, 0.02 to 0.5M of bicine, and 0.02 to 0.5M of sodium chloride, then reacting 24 μg of TRAIL protein and a precipitation solution containing 20 to 30% of PEG MME550, 10 to 100mM bicine, and 5 to 20mM of cadmium chloride. The three-dimensional structure of TRAIL protein consists of an inner sheet comprising strand A, A'', H, C and H; an outer sheet comprising strand B', B, G, D and E; and a loop which links ten strands.

Abstract translation: 目的：提供一种生产TNF相关凋亡诱导配体（TRAIL）蛋白和TRAIL蛋白结晶及其三维结构的方法，从而具有改善活性的TRAIL蛋白，特异性杀死癌细胞和被病毒感染的细胞， 可以生产。 构成：通过以下步骤制备序列号为2的TRAIL：将人TRAIL蛋白质基因插入质粒以产生pET3a-TLS; 用pET3a-TLS转化大肠杆菌以产生大肠杆菌BL21-TLS（pET3a-TLS）（KCCM-10169）; 在22至37℃下孵育转化的大肠杆菌BL21-TLS（pET3a-TLS）（KCCM-10169） 并在孵育过程中加入IPTG以表达TRAIL基因; 破碎细胞，收集不溶性片段，使片段变性和重折叠以收集可溶性蛋白质; 用阳离子交换色谱法和尺寸除去色谱法处理可溶性片段和可溶性蛋白质以分离和纯化TRAIL蛋白质。 通过使用含有20〜40％的PEG MME550，0.02〜0.5M的二胺和0.02〜0.5M的氯化钠的溶液使TRAIL蛋白质结晶，然后使24μg的TRAIL蛋白质和含有20〜30％ 的PEG MME550,10至100mM的二碳酸和5至20mM的氯化镉。 TRAIL蛋白的三维结构由包含链A，A“，H，C和H的内层组成; 包含股线B'，B，G，D和E的外部片材; 以及连接十条线的环路。

公开(公告)号：KR1020020056565A

公开(公告)日：2002-07-10

申请号：KR1020000085947

申请日：2000-12-29

Applicant: 동화약품주식회사 ,  학교법인 포항공과대학교

Inventor： 정용호 ,  유제만 ,  황연하 ,  윤제인 ,  오병하 ,  차선신 ,  임경묵

IPC: C12N15/63

CPC classification number: C07K14/70575 ,  C07K2299/00

Abstract: PURPOSE: Provided are a three-dimensional structure of TRAIL-sDR5 complex of a human derived apoptotic factor and a receptor thereof by X-ray crystallography and TRAIL(TNF Related Apoptosis Inducing Ligand) deletion mutant protein. CONSTITUTION: TRAIL-sDR5 complex is prepared by mixing TRAIL protein of SEQ ID NO:1 and sDR5 protein of SEQ ID NO:2. It can be crystallization method comprises the steps of: injecting a solution consisting of 16% of polyethyleneglycol 3000, 0.05M of sodium acetate(pH 4.5), 0.22 M of sodium acetate and 0.6M of sodium chloride into a well; mixing the precipitation solution and protein solution in a mixing ratio of 1:1 on the surface of a cover slip; and covering the well with the cover slip to crystallize the protein.

Abstract translation: 目的：提供通过X射线晶体学和TRAIL（TNF相关凋亡诱导配体）缺失突变蛋白的人源性凋亡因子及其受体的TRAIL-sDR5复合物的三维结构。 构成：通过混合SEQ ID NO：1的TRAIL蛋白和SEQ ID NO：2的sDR5蛋白来制备TRAIL-sDR5复合物。 结晶方法包括以下步骤：将由16％聚乙二醇3000,0.05M乙酸钠（pH4.5），0.22M乙酸钠和0.6M氯化钠组成的溶液注入孔中; 将沉淀溶液和蛋白质溶液以1：1的混合比混合在盖板的表面上; 并用盖子覆盖井，使蛋白质结晶。