Overexpression and refolding of thioredoxin/TRAIL fusion from inclusion bodies and further purification of TRAIL after cleavage by enteropeptidase

被引：35

作者：

Gasparian, Marine E. ^{[1
]}

Ostapchenko, Valeriy G.

Yagolovich, Anne V.

Tsygannik, Igor N.

Chernyak, Boris V.

Dolgikh, Dmitry A.

Kirpichnikov, Mikhail P.

机构：

[1] Lab Xray Struct Anal, Shemyakin, Russia

[2] RAS, Ovchinnikov Inst Bioorg Chem, Moscow 117997, Russia

[3] Moscow MV Lomonosov State Univ, AN Belozersky Inst Phys Chem Biol, Moscow 119899, Russia

来源：

BIOTECHNOLOGY LETTERS | 2007年 / 29卷 / 10期

基金：

俄罗斯基础研究基金会;

关键词：

apoptosis; enteropeptidase; protein refolding; thioredoxin fusion; TRAIL;

D O I：

10.1007/s10529-007-9446-y

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 [微生物学]; 0836 [生物工程]; 090102 [作物遗传育种]; 100705 [微生物与生化药学];

摘要：

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 angstrom, c = 141.5 angstrom. Apoptosis induced in HeLa cells by purified TRAIL was 5-fold enhanced by emetine.

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页码：1567 / 1573

页数：7

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