Recombinant prokaryotic plasmid construction and high expression of FUS1 gene.
- Author:
Bao ZHANG
1
;
Xia HUO
;
Lin PENG
;
Zong-li QI
;
Xi-jin XU
;
Yan LI
;
Bo QIU
;
Liang-kai ZHENG
Author Information
- Publication Type:Journal Article
- MeSH: Blotting, Western; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Escherichia coli; genetics; Humans; Mesenchymal Stromal Cells; cytology; metabolism; Plasmids; genetics; Recombinant Proteins; biosynthesis; Transformation, Genetic; Tumor Suppressor Proteins; biosynthesis; genetics; Umbilical Cord; cytology
- From: Journal of Southern Medical University 2007;27(5):638-640
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct the prokaryotic plasmid of FUS1 gene for efficient FUS1 expression in E.coli strain Rosetta(DE3)2plys.
METHODSThe full-length FUS1 gene was amplified by PCR from the total RNA of umbilical mesenchymal stem cells and cloned into pET-32a(+) vector followed by identification with PCR and sequencing. The recombinant plasmid pET-32a(+)-FUS1 was transformed into the E.coli strain Rosetta(DE3)2plys and the target protein expression was induced by IPTG.
RESULTSThe plasmid pET-32a(+)-FUS1 was obtained successfully as verified by PCR and sequence analysis. High expression of the fused FUS1 protein was achieved after induction by low-concentration IPTG (25 micromol/L) for 3 h, and the recombinant FUS1 protein accounted for 40% of the total bacterial protein of Rosetta(DE3)2plys.
CONCLUSIONThe recombinant FUS1 plasmid has been successfully cloned, which allows highly efficient FUS1 expression in Rosetta (DE3)2 plys.
