Expression of Human
Trefoil Factor 3 in Pichia pastoris and Its
Biological Activity Analysis
WANG Yan-Ru, REN Hong-Wei, AN Lin, FANG Min, LI Ling-Yuan,
RU Bing-Gen*
( National Laboratory of Protein Engineering, College of Life Science,
Peking University, Beijing 100871, China )
Abstract 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 AOX1 promoter and
α-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.
Key words trefoil factor 3； Pichia pastoris； ferment； expression
*Corresponding author： Tel, 86-10-62751842； Fax, 86-10-62751842； e-mail, [email protected]