Activity Anatysis And Prokaryotic Expression Optimization Antifungal Peptide MAF-1 From Musca Domestica

Objective:To optimize the prokaryotic expression system of recombinant p ET-28a(+)-MAF-1(Musca domestica antifungal peptide-1).Method:1.The expression system of the recombinant MAF-1were constructed with Escherichia coli BL21 and E.coli Origami B, expression levels of which were analyzed under different culture conditions, including changes of culture temperature, time and concentration of IPTG. The expression recombinant MAF-1 was purified by nickel ion metal chelating agent affinity chromatography and detected using SDS-PAGE and Quantity One gel electrophoresis image analysis system. 2. Recombinant MAF-1 antifungal activity in vitro and minimum inhibitory concentration were tested by microdilution test using Candida albicans ATCC10231 as indicator bacteria,We observed the antifungal activity of the expressive recombinant MAF-1 after treatment with trypsin, frozen-thaw and heat stress, and detected its hemolytic activity in vitro and agglutination activity. And the antifungal activity of the MAF-1 with or without His-tag was also tested.3. We constructed fusion gene p ET-28a(+)-MAF-1-LZM(lysozyme of Musca domestica)and its E.coli Origami B/(DE3) expression system using synchronous enzyme technology. The antifungal activity of the fusion gen expression product was detected. Results:1.Based on the expression system of the recombinant plasmid PET-28a(+)-MAF-1 and E.coli Origami B/(DE3), the the gray value of recombinant MAF-1 pre-purification and purification were respectively: 808±4,412±2, and the concentration of the purified recombinant MAF-1 was 0.706 mg/ml, under the culture condition of 34℃, IPTG of 0.025mmol/L, 18 hours, while the gray value and concentration were 258±5,173±7 and 0.401mg/ml respectively, based on E.coli BL21/(DE3) under the culture condition of 37℃, IPTG of 1.0mmol/L, 6 hours. The result showed a statistical significance(p<0.05). 2. The minimum inhibitory concentration of recombinant MAF-1from the supernatant of E.coli Origami B/(DE3) expression system was 70μg/ml using the Candida albicans as tested bacteria, but increased to 100μg/ml of that from the inclusion body of E.coli BL21/(DE3) expression system.Recombinant MAF-1 did not have hemolytic activity and hemagglutinating activities. Its antifungal activity disappeared after treated with trypsin, while the activity can be maintained when treated with heat stress of 95℃ and frozen-thaw of-80℃. 3. The recombinant MAF-1-LMZ fusion protein was successfully expressed in E.coli Origmi B/DE3 and the purified target protein had antifungal activity and its inhibitory concentration was 100μg/ml. Conclusion: 1. Using E.coli Origmi B/(DE3), there was a higher soluble expression level of recombinant MAF-1 than that using E.coli BL21/(DE3),and the highest expression level under the culture condition of 34℃, IPTG of 0.025mmol/L, 18 hours. 2. There was a higher antifungal activity of recombinant MAF-1from the E.coli Origmi B/(DE3) expression system than E.coli BL21/(DE3).3.we constructed the fusion gen of recombinant MAF-1-LMZ fusion protein which was successfully expressed in E.coli Origmi B/DE3 and showed an antifungal activity. But MAF-1-LMZ did not improve antifungal activity of MAF-1.The results should support further researches of MAF-1.