High-efficiency Expression And Purification In Escherichia Coli To Obtain Nonfusion Melittin

Melittin（MET）is a kind of polypeptide substance,accounting for about 50%of the total bee venom.Due to its broad spectrum and high antibacterial activity and low drug resistance,it has a good application prospect.Because melittin is highly lethal to cells and easily degraded by proteases in bacteria,melittin often needs to be expressed with other label proteins to neutralize its innate toxicity and obtain its recombinant proteins.Most protein tags have the problem of amino acid residues after digestion.In order to obtain polypeptides with the same structure as natural melittin through recombinant expression and purification methods,this study fused the melittin gene with different tag genes to overcome the lethal effect of melittin on host cells,and screened out recombinant strains with high soluble expression.Melittin was purified by reversible phase transformation cycle,affinity chromatography,protease digestion and molecular sieve chromatography,and its antibacterial activity was confirmed by antibacterial experiments.The main research contents are as follows:（1）Recombinant plasmids fused with His or GST,MET and tag SUMO were constructed and transferred into Escherichia coli BL21（DE3）to achieve the expression and purification of the fusion proteins His-SUMO-MET and GST-SUMO-MET.SUMO label and MET were reconstructed into p ET-28a and pGEX-6p-1 vectors,and then transformed into Escherichia coli BL21（DE3）receptor cells to achieve soluble expression of fusion proteins His-SUMO-MET and GST-SUMO-MET.The fusion proteins His-SUMO-MET and GST-SUMO-MET were purified by the supernatant of the cell breakdown of the recombinant bacteria with the yield of 2.2 mg and 36.7mg per liter of fermentation broth,respectively.（2）E.coli BL21（DE3）/pGEX-6p-1-Ulp1,a recombinant expression strain of the tool enzyme GST-Ulp1,was purified by GST column to obtain the tool enzyme GST-Ulp1.The GST column combined with the fusion protein GST-SUMO-MET was added and digested overnight at 4℃.The enzyme digestion products were purified by Superdex Peptide 10/300 GL column chromatography to obtain s MET with purity≥90%and 1.1 mg s MET per liter of fermentation solution.（3）Recombinant E.coli BL21（DE3）/p ET21-ELP-Intein-MET was constructed,and the soluble fusion protein ELP-Intein-MET was obtained after induced expression.The purification conditions of fusion protein ITC were optimized.The optimal conditions were equal volume mixing of 6 mol·L^-1Na Cl solution and heating at 40℃for 10 mins.The optimum conditions for self-cleavage of fusion protein were p H 7.0 and 30℃for 30 h.e MET with 92%purity was purified by Superdex Peptide 10/300 GL gel column.2.7 mg e MET was purified per liter of fermentation liquid.（4）E.coli JM109,Bacillus subtilis 168 and Staphylococcus aureus were used as test strains.The results of the Oxincup method showed that the purified melittin s MET and e MET showed obvious inhibitory effects on the three test strains.The antibacterial activity for S.aureus was greater than that for B.subtilis 168 than that for E.coli JM109.s MET showed stronger antibacterial activity than e MET.The microdilution coating method showed similar bacteriostatic results.Finally,the minimum bacteriostatic concentrations of s MET and e MET were determined.The minimum inhibitory concentrations in S.aureus,B.subtilis 168 and E.coli JM109 were 12.5μg·m L^-1,50μg·m L^-1and 100μg·m L^-1and 128μg·m L^-1,256μg·m L^-1and 512μg·m L^-1,respectively.