Abstract:The preparation of melittin by biological method is a key technology for the large-scale preparation of melittin. To achieve efficient expression of melittin, the melittin gene met coding mature peptide and its promet containing the signal peptide, leader peptide and mature peptide were chemically synthesized. The two genes were linked with the maltose binding protein gene mbp and cloned into the vector pET15-b to construct the recombinant plasmid pET-MBP-MET and pET-MBP-proMET. The recombinant plasmids were then transformed into Escherichia coli BL21 (DE3) and induced with 0.1 mmol/L IPTG. The fusion protein MBP-MET and MBP-proMET were efficiently expressed in E. coli in soluble form treated overnight at 30 ℃. The two soluble target proteins were purified by two steps of Ni-affinity and Dextrin Sepharose HP affinity. The fusion proteins MBP-MET and MBP-proMET were obtained with an over 90% purity. About 20 mg of pure protein was prepared by 1 L of recombinant E. coli culture. The inhibition experiments of bacteria by MBP-MET and MBP-proMET were carried out. The obvious inhibition zone was observed in the Oxford Cup antibacterial experiments. The minimum inhibitory concentrations of MBP-MET and MBP-proMET were about 100 μg/mL and 140 μg/mL, respectively. Highly efficient expression was successfully achieved in this research. The function verification of melittin was confirmed, providing an efficient and safe synthetic route for the biological preparation of melittin.
