High-level expression and secretion of recombinant mouse endostatin by Escherichia coli.
The expression of murine endostatin was achieved by placing its gene downstream of an alkaline phosphatase gene (phoA) promoter. To ensure proper folding and secretion of the recombinant protein, the mouse endostatin was fused with alkaline phosphatase signal peptide. SDS/polyacrylamide gel electrophoresis analysis of the culture medium of recombinant Escherichia coli cells revealed that endostatin was efficiently secreted. The signal peptide was efficiently cleaved during secretion as demonstrated by N-terminal amino acid sequencing. The maximum yield of secreted endostatin during fermentation was 40 mg/liter. Up to 28 mg of endostatin was purified from 1 liter of cell culture broth. The biological activity of recombinant protein was tested in a bovine aortic endothelial (BAE) cell proliferation assay. The recombinant endostatin inhibited the growth of BAE cells stimulated by basic fibroblast growth factor, and its ED50 was comparable to that from a previous report. Flow cytometric measurements of BAE cells cultivated in medium with endostatin demonstrated a cell cycle arrest mainly in the G0/G1 phase and a decrease in the S phase.
Xu
Ren
R
Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Sciences, 320 Yue Yang Road, ShangHai 200031, P.R.China.
Du
Peng
P
Fan
Jin-Jiang
JJ
Zhang
Qian
Q
Li
Tsai-Ping
TP
Gan
Ren-Bao
RB
D000251:Adenosine Triphosphatases / Q000235:genetics; D000818:Animals; D002417:Cattle; D002453:Cell Cycle; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics*; D043169:Endostatins; D004730:Endothelium, Vascular; D004926:Escherichia coli; D051379:Mice; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020539:Sequence Analysis, Protein
D043169:Endostatins; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D003094:Collagen; D000251:Adenosine Triphosphatases