High-efficiency Fusion Expression And Purification Of Melittin In Escherichia Coli BL21(DE3)

Melittin is an amphiphilic?-helical active short peptide with various physiological activities such as antibacterial,anti-inflammatory,anti-tumor,anti-viral and hemolysis.It has broad application prospects in the pharmaceutical,health care products and cosmetics industries.But melittin is difficult to be expressed and purified in microbials since its anti-bacterial activities and small molecular weight.In this study,the melittin realized fusion expression through the fusion of its gene with the different solubilizing-tag genes to overcomes the lethality of melittin to host cells.The recombinant strains with highly soluble expressed melittin were screened.The melittin was purified by affinity chromatography,protease digestion and molecular sieve chromatography.And its antibacterial activity was confirmed by antibacteria test.The main research contents include:?1?The melittin propeptide gene containing a signal peptide,a leader peptide and a mature peptide was chemically synthesized.Primers were designed and the proMET gene,the mature peptide MET gene were cloned.They were fused with the different tags of 6×His,Strep II,MBP and GST,and induced expression in E.coli BL21.The soluble expression of melittin was achieved in recombinant E.coli BL21/pET-MBP-proMET,E.coli BL21/pET-MBP-MET and E.coli BL21/pGEX-MET under the induction condition of 0.1 mmol·L^-1 IPTG.?2?The soluble proteins of the recombinant E.coli BL21/pET-MBP-proMET and E.coli BL21/pET-MBP-MET were firstly purified by Ni-affinity chromatography and followed by MBP-affinity chromatography.The target proteins were obtained with a purity of over 90%.About 20 mg of the purified fusion proteins MBP-proMET and MBP-MET can be produced per1 L of bacterial culture.Then,MBP-proMET and MBP-MET were digested with TEV protease.However,three bands of MBP-proMET,MBP and proMET were observed in Tricine-SDS-PAGE gels and they could not be completely separated with Superdex 75chromatography.To optimize isolation,the HRV 3C protease cleavage site was introduced,or flexible linkers"GG"or"GGGG"were introduced at the C-terminal of the TEV protease cleavage site,but the target bands proMET and MET could not be accurately separated.?3?GST-MET was solubly expressed in E.coli BL21/pGEX-MET,and the partial GST tag proteins were over-expressed.The recombinant protein GST-MET was purified by GST-affinity chromatography.The results showed that GST was also enriched and mixed with the purified GST-MET.The proteins GST and GST-MET were digested with PreScission protease for 12 h and purified by two-step chromatography using Glutathione Sepharose HP and Superdex Peptide column.The protein MET with a purity of over 90%was obtained.?4?The antibacterial activity of MET was confirmed by the Oxford Cup bacteriostatic test and the microdilution coating test.The results showed purified recombinant MET has obvious antibacterial effects against E.coli JM109,Bacillus pumilus and Staphylococcus pasteuri.The growth inhibition rates against Gram-positive strains B.pumilus and S.pasteuri were 97%and68.6%,respectively,which was higher than 52.5%against Gram-negative strain E.coli JM109.The tertiary structure of MET was predicted by the I-TASSER server and aligned with the reported melittin structure?PDB ID:2MLT|A?using PyMOL,RMSD=1.779?.