We described construction of a novel vector, pAOX-HT, for direct cloning of polymerase chain reaction (PCR) amplified fragments and expression in Pichia pastoris. The pAOX-HT serves both as a T-vector and expression vector and can be generated from a parent plasmid by digestion with restriction enzyme XcmI. To minimize the non-recombinant background, the parent vector pAOX-HT-BSK was designed to contain a large insert. The cloning efficiency was above 95% when tested with PCR products. The linearized pAOX-HT was engineered to harbor a potential AflII site (CT) upstream of the T/A cloning site. AnAflII site was reconstructed when the PCR product with 5’-TAAG sequence was ligated into the T/A cloning site. Taking advantage of this property, we digested the ligated products by restriction enzyme AflII before transformation to eliminate the clones containing inserts with undesired orientation. By using pAOX-HT vector, the lipase B gene from Candida Antarctica was efficiently cloned and expressed in P. pastoris and the recombinant protein was purified by affinity chromatography. These results demonstrate that the pAOX-HT might serve as a useful tool for gene function study.
Key words: T vector, expression vector, yeast expression, restriction endonuclease digestion.
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