Buthus Martensii Anti-nerve Excitability Peptide Expression, Purification And Some Properties

Multiple alignments show ANEP is homologic with depressant insect neurotoxin, and it belongs to this family, they all affect Na~+ channel. By WHAT-IF, we forecasted three-dimensional structure and disulfide linkage of ANEP, whose structure is similar with most large chain neutoxins, is mainly composed of one a helix and three anti-parallel 6 sheets. These components construct compact nucleus, it is conservative disulfide linkage partner mode that preserves this spacial structure. Disulfide linkage can't form when ANEP is expressed alone in E.coli, insert it's coding sequence into pSYPU-1b, ANEP consists in supernatant of cytoplast of E.coli.We processed one-step purification of ANEP in Chelating Sepharose Fast Flow by His tag. By changing different conditions of elution, we can get electrophoresis pure sample. Combined with spacial structure of ANEP, under natural conditions and strong denaturant was added, we investigated behavior of chromatography when His tag was placed at different end of peptide. We found spacial shielding effect purification notably, when His tag is placed at C end of ANEP, His tag is shown completely, and peptide is absorbed at the column tightly. By optimizing eluting condition, we could get about 3mg peptide from one litre Exoli. But when His tag was located at N end, we could just get 0.8mg.We investigated the rule of protein expression that under control of T7 lacUV5 promoter when IPTG and lactose were used as inducer. In virtue of SDS-PAGE analytical method and E.coli growing circumstance, we investigated inducing time , inducer concentration and abduction length which affected the expression of recombined protein, and glucose inhibiting effect affected protein expression when lactose was used as inducer. Glucose inhibiting effect is very notable in the experiment, lactose is just added after metaphase of log, and we can observe little protein is expressed.Plasmid stability experiment showed the vegetal ability of recombined E.coli wasn't weak, recombinant was inoculated again after 12 hours cultivation under resistive oppression, after 20 times, the imitative rate of plasmid is 0317‰. And the ability of...