[Expression of human intestinal trefoil factor in Pichia pastoris and its biological activity on intestinal epithelium in vitro].
In order to produce relatively large amounts of recombinant human intestinal trefoil factor and assess its biological activity. The expression plasmid pPIC9-hITF containing AOX1 promotor and the sequences of secreting signal peptides was transformed into the yeast cells. Then through selection, positive transformants were cultivated in fermentation basal salts medium in a 5L fermenter to obtain large amount product with low cost. The secreted peptides were then purified by a combination of ionic exchange chromatography and molecular sieve. To verify the product, electrospray mass spectrometry analyses was used to determine the structure of rhITF and Western Blotting was performed to test the immunological activity. Furthermore, the biological activity of the peptide was examined by experiments from cell to tissue. The nucleotide sequence of rhITF was the same as expected. With a 5-L fermenter, 253mg of hITF was isolated at the purity of 96% from 3.5 L of yeast fermentation broth. The expression level for recombinant human ITF in this yeast system was 73.33mg/L. In our study, we provided a way to gain a production among milligram to gram of recombinant human ITF by the use of a yeast expression system. As human ITF are difficult to purify in any significant amount from tissue extraction, the way described may become a valuable tool in obtaining pure peptide for further studies of trefoil peptide function.
Shanghai Ruijin Hospital, Shanghai Jiaotong University, Shanghai 200025, China.
Wei
W
Liu
Hua-Bao
HB
Zheng
Xue-Mei
XM
Zhong
Sheng
S
Yang
Chun-Di
CD
Xu
D002465:Cell Movement / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D005285:Fermentation; D006801:Humans; D007421:Intestine, Small / Q000166:cytology; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology*; D000071164:Trefoil Factor-2
D010455:Peptides; D011994:Recombinant Proteins; D000071164:Trefoil Factor-2