Site Directed Modify Capra Hircus Lysosomal Alpha-mannosidase To Decreased Its Sensetivity To SW

Swainsonine, an alkaloid with significant physiological activity, is an important lysosoaml α-mannosidase inhibitor, mainly from locoweed; Consumption of these plants by grazing animals leads to a chronic neurologic disease similar to alpha-mannosidosis. There is currently no effective antidote for locoweed poisoning in clinical use either domestically in China or abroad. With a view to the research of Capra hircas LAM, in order to solve the problem of locoweed toxocity. In our prophase research, the chLAM(Capra hircas LAM) cDNA was cloned by RT-PCR((accession no. JN602369)). Bio-informatics analysis found the chLAM, an overall structural model of chLAM included five peptides A-E. The active pocket characterized by 9 amino acids residues are conserved between species’ LAM. After inspecting the difference binding modes of substrate man5 and inhibitor SW with wild type chLAM,it was predicated that if Trp A28 and Tyr D58 of wild type of chLAM mutate to Gly, the chLAM may increase resistance to SW, in the meantime, the binding affinity of wild type chLAM with man5 does not appear to be significantly different. In this study, the definite mutation chLAM gene was obtained from the full-length chLAM gene by overlap extension PCR, and truncate E peptide, the truncated and mutant gene was inserted into expression vector pPICZαA to construct Pichia yeast X33 expression system. After inducing it to express with methanol, we obtained the expressed product, then, verified the product. The research mainly focuses on the following aspects:1. Based on the 3D structure of chLAM from bioinformation analysis, Trp A28 and Tyr D58 were chosen as the mutation sites. They both were chosen to mutate into Gly. The overlap extension PCR was used to obtain the mutant gene sequence chLAM-A28-D58, then, we truncated E peptide from the chLAM-A28-D58, finally obtain the truncated and mutant gene chT-A28-D58.2. The encoding sequences of the full-length and chT-A28-D58 gene were connected to the Pichia yeast expression vector pPICZαA with 6×His tag to obtain recombinant plasmid pPICZαA-chLAM and pPICZαA-chT-A28-D58, and two recombinant plasmids were electrotransformated into Pichia yeast X33. After inducing by 1% methanol for 120 h, expressed product were subject to ulatrasonication and collected supernatant. We found the target protein from the supernatant, SDS-PAGE showed the the full-length protein chLAM with a MW of approximately 107 KDa and the chT-A28-D58 protein MW is 87 kDa.3. The target protein was purified by Ni-NTA affinity chromatography, a series of elution fractions were collected to analyzed by 8% SDS-PAGE. A clear single target band was found in elute with 100 mM imidazole. The western blot of purified target protein showed about 107 kDa and 87 kDa bands and displayed the strong positive reaction. The result indicate the chLAM and chT-A28-D58 gene were successfully expressed in Pichia yeast X33.4.The optimum temperature and pH of chLAM were 55 ℃and 6.0, while that of the chT-A28-D58 were 45 ℃and 6.0, respectively. Zn2+、Ca2+、Mg2+ and Cr3+ with the concentration fo 5-10 mM had certain promoting effect to enzyme activity and EDTA、Al3+、Co2+ could inhibit the enzyme activity. Added with SW, activity of chT-A28-D58 decreased by 42.7%, while the activity of chLAM decreased by 73.5%. The decline rate of chT-A28-D58 was significantly higher than chLAM.