蜂毒肽在大肠杆菌中的高效融合表达与纯化

High-Efficiency Fusion Expression of Melittin in Escherichia coli and Its Purification

生物法制备蜂毒肽是实现大量制备蜂毒肽的关键技术。为实现蜂毒肽的高效表达,通过化学法合成含信号肽、前导肽和成熟肽的蜂毒肽基因promet和蜂毒肽成熟肽基因met,与麦芽糖结合蛋白基因mbp连接,克隆到载体pET15-b,构建重组质粒pET-MBP-MET和pET-MBPproMET。重组质粒转化大肠杆菌Escherichia coli BL21(DE3),以0.1 mmol/L IPTG进行诱导,30℃下过夜诱导,融合蛋白MBP-MET、MBP-proMET在大肠杆菌中以可溶性形式高效表达。两种目标蛋白质经Ni柱和Dextrin Sepharose HP两步亲和纯化,得到了纯度达90%以上的融合蛋白MBP-MET、MBP-proMET,培养1 L大肠杆菌菌液能制备约20 mg纯蛋白质。通过牛津杯抑菌实验发现,MBP-MET、MBP-proMET能明显抑制细菌生长,产生明显的抑菌圈,MBP-MET和MBP-proMET最低抑菌浓度(MIC)分别为100μg/mL和140μg/mL。本研究首次实现了蜂毒肽的高效表达,并进行了抑菌功能验证,为生物法制备蜂毒肽提供了高效、安全的合成途径。

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 p ET-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 100μg/mL and 140μg/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.