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 IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R-IFNα2b 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.

[1]  D. Botstein,et al.  Plasmid construction by homologous recombination in yeast. , 1987, Gene.

[2]  J. Sambrook,et al.  Molecular cloning–A laboratory manual. New York: Cold Spring Harbor Laboratory. 1982, 545 S., 42 $ , 1985 .

[3]  F. Ruddle,et al.  Direct cloning of genomic DNA by recombinogenic targeting method using a yeast-bacterial shuttle vector, pClasper. , 1999, Genomics.

[4]  A. Shinohara,et al.  Homologous recombination and the roles of double-strand breaks. , 1995, Trends in biochemical sciences.

[5]  R. S. Muir,et al.  Gene disruption with PCR products in Saccharomyces cerevisiae. , 1995, Gene.

[6]  F. Prado,et al.  New in-vivo cloning methods by homologous recombination in yeast , 1994, Current Genetics.

[7]  K. Kitagawa,et al.  In vivo site-directed mutagenesis of yeast plasmids using a three-fragment homologous recombination system. , 2002, BioTechniques.

[8]  K. Oldenburg,et al.  Recombination-mediated PCR-directed plasmid construction in vivo in yeast. , 1997, Nucleic acids research.

[9]  D. Botstein,et al.  Genetic properties of chromosomally integrated 2μ plasmid DNA in yeast , 1982, Cell.

[10]  J. Daniel.  DNA insertion system for complex yeast shuttle vectors , 1995, Current Genetics.

[11]  J. Haber,et al.  Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae , 1999, Microbiology and Molecular Biology Reviews.

[12]  A. Willems,et al.  Studies on the transformation of intact yeast cells by the LiAc/SS‐DNA/PEG procedure , 1995, Yeast.

[13]  M. Labouesse,et al.  A family of low and high copy replicative, integrative and single‐stranded S. cerevisiae/E. coli shuttle vectors , 1991, Yeast.

[14]  J. Broach,et al.  6 Circular DNA Plasmids of Yeasts , 1991 .

[15]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[16]  A. Adams,et al.  Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual , 1998 .

[17]  P. Sung,et al.  Recombination factors of Saccharomyces cerevisiae. , 2000, Mutation research.

[18]  P. Philippsen,et al.  New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae , 1994, Yeast.

[19]  M. Pall,et al.  A series of yeast shuttle vectors for expression of cDNAs and other DNA sequences , 1993, Yeast.

[20]  P Manivasakam,et al.  Micro-homology mediated PCR targeting in Saccharomyces cerevisiae. , 1995, Nucleic acids research.

[21]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[22]  M. Resnick,et al.  In vivo site-directed mutagenesis using oligonucleotides , 2001, Nature Biotechnology.

[23]  Y Luo,et al.  Minimum length of sequence homology required for in vivo cloning by homologous recombination in yeast. , 1997, Plasmid.

[24]  E. Degryse,et al.  In vivo cloning by homologous recombination in yeast using a two‐plasmid‐based system , 1995, Yeast.

[25]  M. Kupiec.  Damage-induced recombination in the yeast Saccharomyces cerevisiae. , 2000, Mutation research.

[26]  O. Ozier-Kalogeropoulos,et al.  A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. , 1993, Nucleic acids research.