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An Improved Strategy for High‐Level Production of Human Vasostatin120–180
Author(s) -
Sun QiMing,
Chen LiLi,
Cao Lin,
Fang Lei,
Chen Cheng,
Hua ZiChun
Publication year - 2008
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp049583x
Subject(s) - affinity chromatography , enteropeptidase , chemistry , fusion protein , ammonium sulfate precipitation , western blot , sephadex , escherichia coli , yield (engineering) , chromatography , expression vector , biochemistry , microbiology and biotechnology , recombinant dna , biology , gene , size exclusion chromatography , enzyme , materials science , metallurgy
We previously reported a strategy for expression and purification of human Vasostatin120–180 (VAS), a potent angiogenesis inhibitor in a GST fusion form; however, the yield of 7.2 mg per liter of culture was relatively low. The aim of this study was to develop a more efficient system to improve and facilitate the production of VAS protein in a soluble and native form in Escherichia coli . The VAS gene with optimized condons was cloned into pET28a and overexpressed as a N‐terminal His‐tagged fusion protein. Between His‐tag and VAS, an enterokinase recognition site was introduced to release the intact VAS. Optimal expression of soluble His‐VAS was achieved by examining the contribution of chaperone coexpression and lower temperature fermentation. Ammonium sulfate precipitation was first employed to remove nucleic acid and partial host proteins. When further purified by Ni 2+ affinity chromatography, 40 mg of His‐VAS was isolated with purity over 85% from 1 L of culture. After desalting with Sephadex G15 and digestion with His‐EK, followed by the removal of the His‐tag and His‐EK with Ni 2+ ‐NTA resin, 21 mg of intact VAS was finally obtained from 1 L of bacterial culture, which was approximately 3‐fold the yield we previously obtained via GST fusion expression strategy. The identity of His‐VAS and VAS was confirmed by Western blot. Purified VAS displayed distinct anti‐angiogenic activity, which was shown by the endothelial cell proliferation inhibition assay and chicken chorioallantoic membrane assay. In sum, we greatly improved the yield of intact and bioactive VAS protein, and using this successful example, we propose a more efficient system for the high‐level production of intact functional proteins, especially for low molecule weight peptides.