Expression And Characterization Of A Truncated α-mannosidase From Capra Hi Reus Lysosomal In Pichia Pastoris

α-Mannosidase (AMA) belongs to the glycoside hydrolyzing enzymes family. VariousAMA widely distributed in animals, plants and microorganisms are involved in thebiosynthesis and turnover of N-linked glycoproteins process. Locoweeds are main poisoningplants on the western lands in China. In addition to the destruction of grassland ecosystem, ithas causing goats, horses, and sheep death and large economical loss every year. The plantscontaining the toxic indolizidine alkaloid swainsonine (SW) that inhibits AMA, and therebyresulting in animal poisoning, eventually death. At present, it has been reported a variety ofanimal’ AMA gene sequences. The mutations in the MAN2B1(LAMAN) gene encodinglysosomal α-mannosidase could cause alpha-mannosidosis and incurable dyserythropoieticanemiaⅡ. The whole open reading frame of the chLAM(Capra hircas LAM, chLAM) cDNAsequence were obtained(accession no. JN602369). In goat, the107.56KDa peptide is furtherpartially proteolysed into three more peptides that are joined by disulfide bridges–constituting all together five peptides (a-e). Thus, our study has focused on truncating thefull-length chLAM based on the three-dimensional structure and the core catalytic region ofα–mannosidase from chLAM and heterologous expression of the truncated genes in this hoststrain to compare the enzyme activity and other characteristics with the full-length of chLAMand analysis the mechanism of the enzyme. Results are as follows：1. The truncated chA, chC, chD and chT were amplified by PCR successfully. Byconstruction the yeast expression vector of pPIC9K-chLAMa, pPIC9K-chLAMcpPIC9K-chLAMd and pPIC9K-chLAMt, linearized, electric into the Pichia yeast GS115,1%methanol induced expression, and determination of activity, we detected the chLAMt activityof expression product of was35U, whereas, there is no activity was detected in chLAMa,chLAMc and chLAMd.2. Due to the6*Histidine tag of chLAMt, purification of the secreted chLAMt was easilyachieved in small scale by using the His-bind purification chromatography kit. SDS–PAGE ofthe purified showed that the band about90kDa was on10%polyacrylamide gel.3. The western blot result indicated that the protein of chLAMt about90kDa wasprobably a native protein of the GS115transformant induced by methanol. 4. chLAMt exhibited an optimal temperature at45-55°C and at pH5.5which is similarpH optima and temperature optima to its full-length protein (chLAM).