Expression And Characterization Of The Truncated D221 Site Of α–mannosidase From Capra Hircus Lysosomal

Locoweeds are main poisoning plants on the western lands in China destructing of grassland ecosystem. The toxic indolizidine alkaloid swainsonine（SW） inhibits AMA resulting in vacuolar due to oligosaccharide accumulation, eventually death. Study has been shown the active center of Capra hircas LAM（chLAM） constituted by 9 conserved amino acid sites. According to the different conservative level of the active site, truncated gene including D221 site or not is named as chLAM-D222 or chLAM-D220 for studying the function of truncation in activity and expression, respectively. Results are as follows:1. According to chLAM gene sequences, the truncated chLAM-D222 and chLAM-D220 are amplified by PCR successfully.2. By construction the yeast expression vector of pPIC9K-chLAM-D222, pPIC9K-chLAM-D220, linearized, electric into the Pichia yeast GS115, 0.6% methanol induced expression, and determination of activity, we detected the activity of protein is 54.7 U, 93.8U and 173.6U and the amount of protein is 12.7 μg/mg、32.4 μg/mg、34.8 μg/mg, respectively.3. Purified recombinant protein in SDS-PAGE reveals a single protein band with the molecular weight of about 108 kDa（chLAM） and 84kDa（chLAM-D222, chLAM-D220）, respectively.4. Purified recombinant protein in western blot, reveals a single antigen band with 108 kDa（chLAM） and 84 kDa（chLAM-D222, chLAM-D220）, respectively.5. The optimal condition of recombinant protein chLAM, chLAM-D222 and chLAM-220 are at 55 ℃, 35 ℃, 35 ℃and at pH 4.5, 3.5, 4.0, respectively. Zn²⁺and Ca²⁺ promote its activity, Cu²⁺ and Co²⁺ inhibit its activity. They are all sensitive to SW; and active truncated protein is more difficult to be inhibited by SW.According to the results, truncation increases the exogenous expression and activity of chLAM in the Pichia yeast.