硫氧还蛋白/TRAIL融合蛋白从包涵体中的过表达与重折叠以及肠肽酶切割后TRAIL的进一步纯化。
Overexpression and refolding of thioredoxin/TRAIL fusion from inclusion bodies and further purification of TRAIL after cleavage by enteropeptidase.
作者信息
Gasparian Marine E, Ostapchenko Valeriy G, Yagolovich Anne V, Tsygannik Igor N, Chernyak Boris V, Dolgikh Dmitry A, Kirpichnikov Mikhail P
机构信息
Laboratory of Protein Engineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 16/10 Miklukho-Maklaya, Moscow, 117997 GSP, Russia.
出版信息
Biotechnol Lett. 2007 Oct;29(10):1567-73. doi: 10.1007/s10529-007-9446-y. Epub 2007 Jul 4.
The human TRAIL gene (encoding residues 114-281) was synthesized by PCR and cloned into plasmid pET-32a. High level expression (1.5 g l(-1)) of thioredoxin/TRAIL fusion was achieved in Escherichia coli strain BL21(DE3), mainly as inclusion bodies. Refolded fusion thioredoxin/TRAIL was cleaved by enteropeptidase and TRAIL was separated from thioredoxin on Ni-NTA agarose. High yield (400 mg l(-1)) of TRAIL without N-terminal methionine and His tag was obtained. Sedimentation coefficient demonstrated that 98% of TRAIL formed trimers. TRAIL formed crystals of space group P3 (1) with unit-cell dimensions a = b = 72.5 A, c = 141.5 A. Apoptosis induced in HeLa cells by purified TRAIL was 5-fold enhanced by emetine.
通过聚合酶链式反应（PCR）合成人TRAIL基因（编码第114至281位氨基酸残基），并将其克隆到质粒pET - 32a中。硫氧还蛋白/TRAIL融合蛋白在大肠杆菌BL21(DE3)菌株中实现了高水平表达（1.5 g l(-1)），主要以包涵体形式存在。经肠肽酶切割重折叠的硫氧还蛋白/TRAIL融合蛋白，TRAIL在镍 - 氮三乙酸琼脂糖上与硫氧还蛋白分离。获得了高产率（400 mg l(-1)）、无N端甲硫氨酸和组氨酸标签的TRAIL。沉降系数表明98%的TRAIL形成三聚体。TRAIL形成了空间群为P3(1)的晶体，晶胞参数a = b = 72.5 Å，c = 141.5 Å。在HeLa细胞中，纯化的TRAIL诱导的凋亡被依米丁增强了5倍。
Zotero 插件