Expression of Human Trefoil Factor 3 in Pichia pastoris and Its Biological Activity Analysis.
The human TFF3 (trefoil factor 3) DNA fragment was amplified by polymerase chain reaction (PCR) from human fetal placenta cDNA. The gene was cloned into the Pichia pastoris expression vector pPIC9K containing AOX 1 promoter and alpha-factor leader sequence. Multi-copies insertion transformants were screened on G418 plates. After the induction by 2% methanol for 48 hours, the expression of dimeric hTFF3 came up to 45% of total proteins in medium, as identified by SDS-PAGE and Western blot assay. The recombinant protein was further purified by S-Sepharose, Q-Sepharose ion-exchange chromatography and Sephacryl-S-100 gel filtration chromatography to the 95% purity, as shown by densitometric scanning. The N-terminus and molecular weight of the recombinant hTFF3 was in good agreement with the native hTFF3. The recombinant protein was proved to have good biological activity of preventing rats from the gastric ulcer induced by hydrochloric acid.
Wang
Y R
YR
National Laboratory of Protein Engineering, College of Life Science, Peking University, Beijing 100871, China. rulab@pku.edu.cn
Ren
H W
HW
An
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L
Fang
M
M
Li
L Y
LY
Ru
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BG