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Construction of a multi-copy Pichia expression vector and expression of human brain-derived neurotrophic factor in Pichia pastoris

Posted by on in 2005
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 [Article in Chinese]
Liang WF, Zhang CC, Yang XC.
Wei Sheng Wu Xue Bao. 2005 Feb;45(1):34-8.
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China. liang1976@hotmail.com

Abstract

A pPICZ alpha1 vector was reconstructed with pPICZ alpha vector and PCR product of His4-Kan sequence from pPIC9K vector. The sequence encoding human brain-derived neurotrophic factor (hBDNF) was inserted into pPICZ alpha1.

The expression vectors carrying 1, 2, 3, 6 copies of expression cassettes were constructed and transformed into Pichia pastoris GS115 respectively. The transformants were screened with G418/Zeocin and induced by 0.5% methanol, and the results of SDS-PAGE confirmed that the expression protein was about 14 kD and the percentage of the protein was about 20% of the total secreted protein.

ELISA and Western blot analysis demonstrated that the protein could be specifically combined with chicken antibodies to human-BDNF.

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