Gasparian Marine E, Ostapchenko Valeriy G, Yagolovich Anne V, Tsygannik Igor N, Chernyak Boris V, Dolgikh Dmitry A, Kirpichnikov Mikhail P
Laboratory of Protein Engineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 16/10 Miklukho-Maklaya, Moscow, 117997 GSP, Russia.
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。
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