title: An improved strategy for high-level production of human vasostatin120-180.
abstract: 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 Ni2+ 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.
authors.affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, College of Life Sciences, Nanjing University, Nanjing 210093, P. R. China.
authors.forename: Qi-Ming
authors.initials: QM
authors.lastname: Sun
authors.forename: Li-Li
authors.initials: LL
authors.lastname: Chen
authors.forename: Lin
authors.initials: L
authors.lastname: Cao
authors.forename: Lei
authors.initials: L
authors.lastname: Fang
authors.forename: Cheng
authors.initials: C
authors.lastname: Chen
authors.forename: Zi-Chun
authors.initials: ZC
authors.lastname: Hua
mesh_terms: D020533:Angiogenesis Inhibitors / Q000235:genetics / Q000494:pharmacology*; D000818:Animals; D037282:Calreticulin / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D002478:Cells, Cultured; D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000187:drug effects; D002846:Chromatography, Affinity / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D004765:Enteropeptidase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
chemical_list: D020533:Angiogenesis Inhibitors; D037282:Calreticulin; D018835:Chaperonin 10; D018834:Chaperonin 60; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C121986:vasostatin; D006639:Histidine; D004765:Enteropeptidase