African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5211

Full Length Research Paper

A novel vector for expression cloning of large numbers of polymerase chain reaction products in Pichia pastoris

Dongming Lan1, Wenkai Wang1, Lijuan Sun1, Yonghua Wang2* and Bo Yang1
  1School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510641, China. 2College of Light Industry and Food Sciences, Key Lab of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou 510641, China.
Email: [email protected]

  •  Published: 04 February 2011



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.