[Construction of a multi-copy Pichia expression vector and expression of human brain-derived neurotrophic factor in Pichia pastoris].
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.
Liang
Wei-feng
WF
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China. liang1976@hotmail.com
Zhang
Chao-chun
CC
Yang
Xi-cai
XC
D019208:Brain-Derived Neurotrophic Factor / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D010843:Pichia / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis*
D019208:Brain-Derived Neurotrophic Factor; D011994:Recombinant Proteins