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Hygromycin‐resistance vectors for gene expression in Pichia pastoris
Author(s) -
Yang Junjie,
Nie Lei,
Chen Biao,
Liu Yingmiao,
Kong Yimeng,
Wang Haibin,
Diao Liuyang
Publication year - 2014
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.3001
Subject(s) - pichia pastoris , biology , selectable marker , hygromycin b , plasmid , multiple cloning site , expression vector , gene , microbiology and biotechnology , expression cassette , green fluorescent protein , recombinant dna , genetics , vector (molecular biology)
Pichia pastoris is a common host organism for heterologous protein expression and metabolic engineering. Zeocin‐, G418‐, nourseothricin‐ and blasticidin‐resistance genes are the only dominant selectable markers currently available for selecting P . pastoris transformants. We describe here new P . pastoris expression vectors that confer a hygromycin resistance base on the Klebsiella pneumoniae hph gene. To demonstrate the application of the vectors for intracellular and secreted protein expression, green fluorescent protein (GFP) and human serum albumin (HSA) were cloned into the vectors and transformed into P . pastoris cells. The resulting strains expressed GFP and HSA constitutively or inducibly. The hygromycin resistance marker was also suitable for post‐transformational vector amplication (PTVA) for obtaining strains with high plasmid copy numbers. A strain with multiple copies of the HSA expression cassette after PTVA had increased HSA expression compared with a strain with a single copy of the plasmid. To demonstrate compatibility of the new vectors with other vectors bearing antibiotic‐resistance genes, P . pastoris was transformed with the Saccharomyces cerevisiae genes GSH1 , GSH2 or SAM2 on plasmids containing genes for resistance to Zeocin, G418 or hygromycin. The resulting strain produced glutathione and S ‐adenosyl‐ l ‐methionine at levels approximately twice those of the parent strain. The new hygromycin‐resistance vectors allow greater flexibility and potential applications in recombinant protein production and other research using P . pastoris . Copyright © 2014 John Wiley & Sons, Ltd.