Expression, purification of human vasostatin120-180 in Escherichia coli, and its anti-angiogenic characterization.
According to codon preference of Escherichia coli, the optimized coding sequence of human vasostatin120-180aa (VAS) was obtained by chemical synthesis and molecular cloning methods. Using PCR and enzyme digestion, the full encoding sequence for VAS was cloned into the E. coli expression vector pALEX and expressed as a GST fusion protein in BL21 (DE3) strain. GST-VAS protein approximately accounted for 45% of the total bacterial proteins. Most of target protein existed in inclusion body. To improve the solubility of GST-VAS, the contribution of low temperature and molecular chaperone co-expression to the solubility of GST-VAS was tested. The results showed that co-expression with chaperons, TF and GroES/GroEL, and low expression temperature cooperatively improved the solubility of GST-VAS from 10 to 85%, and the yield of soluble GST-VAS was sixfold increased. When purified by GST affinity chromatography, 50 mg GST-VAS was obtained with purity over 85% from 1 L culture. Intact VAS was released by enterokinase digestion and further purified by Sephadex G50 gel filtration chromatography. About 7.2 mg intact homogeneous VAS protein was finally produced from 1L bacterial culture. The identity of GST-VAS and VAS was validated by Western blotting analysis. Recombinant VAS protein displayed distinct inhibition of endothelial cell proliferation and anti-angiogenic activity by chick embryo chorioallantoic membrane assay.
Sun
Qi-Ming
QM
The State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, College of Life Sciences, Nanjing University, Nanjing 210093, PR China.
Cao
Lin
L
Fang
Lei
L
Chen
Cheng
C
Dai
Jun
J
Chen
Li-Li
LL
Hua
Zi-Chun
ZC
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D037282:Calreticulin; D049109:Cell Proliferation / Q000187:drug effects; 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; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D051379:Mice; D041681:NIH 3T3 Cells; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012261:Ribonucleoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D012995:Solubility; D013696:Temperature; D014471:Umbilical Veins / Q000166:cytology
D020533:Angiogenesis Inhibitors; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D018835:Chaperonin 10; D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; C121986:vasostatin; D007544:Isopropyl Thiogalactoside; D005982:Glutathione Transferase; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase