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Efficient vectors for expression cloning of large numbers of PCR fragments in P. pastoris
Author(s) -
Rao Ben,
Zhong Xing,
Wang Yaping,
Wu Qiong,
Jiang Zhengbing,
Ma Lixin
Publication year - 2010
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.1753
Subject(s) - pichia pastoris , biology , cloning (programming) , microbiology and biotechnology , plasmid , expression vector , multiple cloning site , restriction enzyme , insert (composites) , recombinant dna , restriction site , orfs , molecular cloning , open reading frame , gene , genetics , complementary dna , peptide sequence , mechanical engineering , computer science , engineering , programming language
The yeast vectors described, pYEV and pYEVB, were designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) and immediate protein expression in Pichia pastoris . The pYEV vector was used to clone PCR fragments obtained by using Taq or similar polymerase mixes, which leave an A‐base overhang. The other vector pYEVB, with the same features for blunt‐end ligation of PCR products, was developed to be complementary to pYEV. These two plasmids were linearized using the restriction enzymes Bfu I and Sch I, respectively. The purified PCR products, without any other treatments, were cloned into the linearized vectors , followed by selection on plates supplemented with X‐gal. Only desired recombinants carrying the target gene in the correct orientation can give typical blue colonies. This screening technique is based on a lac O reconstruction strategy that can produce a full‐length lac O to exhaust endogenous LacI and switch on the transcription of lacZ in the host. The recombinant plasmids extracted from the blue colonies can be linearized by Sal I and transformed into P. pastoris for immediate expression. By using these two vectors, researchers could be saved from the tedious and time‐consuming conventional cloning procedures. Copyright © 2010 John Wiley & Sons, Ltd.