Molecular cloning of squalene synthase gene form Paris polyphylla and its expression in Escherichia coli.
- Author:
Fei GAO
1
;
Xiao-Peng LUO
;
Liang TAO
;
Cheng-Lei LI
;
Chun-Bang DING
;
Hui CHEN
;
Qi WU
Author Information
- Publication Type:Journal Article
- MeSH: Cloning, Molecular; Escherichia coli; genetics; Farnesyl-Diphosphate Farnesyltransferase; genetics; Liliaceae; enzymology; Phylogeny; Recombinant Proteins; biosynthesis
- From: China Journal of Chinese Materia Medica 2013;38(13):2086-2091
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo clone the cDNA sequence of squalene synthase gene from Paris polyphylla, and characterize the biological features of the obtained SQS.
METHODUsing homology cloning and RACE technique, a full-length cDNA sequence of PpSQS gene was isolated from P. polyphylla. The obtained sequence was analyzed by bioinformatics softwares. A plasmid [named pET-30b (+)-PpSQS] was constructed for prokaryotic expression the recombinant PpSQS.
RESULTThe full-length cDNA of PpSQS gene is 1 498 bp, which contains a 1 212 bp ORF. Sequence analysis indicated that PpSQS encoded 403 amino acids residues with a calculated molecular weight (MW) of 46.36 kDa and an isoelectric point (pI) of 6.83. SDS-PAGE results showed that the recombinant PpSQS was expressed in Escherichia coli BL21 (DE3) by inducing with 1 mmol x L(-1) IPTG.
CONCLUSIONThe full-length cDNA sequence of PpSQS gene was obtained from P. polyphylla, and its molecular features were consisted with classic SQS in plant. The recombinant PpSQS was successfully expressed in E. coli.
