Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae.

ABSTRACT

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.