Construction of double-trefoil domain mutant of human trefoil factor 3

Double - trefoil domain mutant of human trefoil factor 3 (hTFF3) was constr ucted through gene engineering and inserted into the pPIC9K plasmid, then e xpressed in the methylotrophic yeast Pichia pastoris. The recombinant prote in was purified through S - Sepharose, Q - Sepharose and Sephacryl S - 100. Molecular weight of the expressed protein was about 12 000 identified by S DS - PAGE and Western - blotting. N - terminal amino acid analyses proved t hat the expressed hTFF3 dimeric form mutant was identical to the native N - terminus. Two protein peaks corresponding to the monomeric and dimeric for m of Di - hTFF3 mutant were identified by the mass spectrometry. The boiact ivity of the mutant was the same as the native dimeric form of hTFF3, and h igher than that of the monomeric mutant.