Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae.
Based on a previously used plasmid pHC11, a new plasmid pHC11R was constructed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R' was co-transformed with a PCR amplified expression cassette of human IFNalpha2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNalpha2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNalpha2b, the expression plasmid pHC11R-IFNalpha2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R-IFNalpha2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.
Chen
Xiangling
X
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China.
Yuan
Hanying
H
He
Wei
W
Hu
Xianghua
X
Lu
Hong
H
Li
Yuyang
Y
D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014162:Transfection
D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins