Study On The Optimization Of Expression Conditions Of Peptide CGA-N46and Purification

CGA-N46is one small peptide and has high antifungal activity to Candiaalbican. So, it has great potential to be an antifungal drug. Gene engineering is oneof efficient method to get CGA-N46. But the expression level is low and thepurification is difficult. In this study, the engineering bacterium Bacillus subtilisDB1342(p-3N46) was used as an experimental strain to research the influencingfactors and expression conditions. At the same time, the purification process wasresearched. One-factor-one-time experiment was used to selecte the basicfermentation medium, the optimal carbon and the optimal nitrogen. The resultsshowed that2×MSR medium was benefit to expresse CGA-N46, dextrin was theoptimal carbon and peptone was the optimal nitrogen. Factorial factors design wasused to select the significant influencing factors. The results showed that carbon andnitrogen were the significant influencing factors for the expression of CGA-N46. Atthe same time, the values of other factors were determined. The value of significantinfluencing factors was determined by the steep hills experiment and the center ofthe combination design experiment. The fermentation medium and conditons wereobtained as follows:16.6g/L of dextrin,19.2g/L of peptone,50g/L of yeast extract,6g/L of K2HPO4·3H2O,6.5of pH,5%of inoculation amount,1h of the inductiontime,56h of fermentation time and30℃of fermentation temperature. Theexpression amount of CGA-N46was4.21times under the optimal fermentationconditions than that of the pre-optimal fermentation conditions. Ammonium sulfateprecipitation was used to separate the target protein from the supernatant bycentrifugation of the fermentation culture.70%ammonium sulfate saturation wasthe suitable saturation to precipitate CGA-N46. The highly pure sample ofCGA-N46was gotten by silica adsorption chromatography and gel exclusionchromatography. The purity of target protein is58.2%. Liquid culture method wasused to test the antifungal activity of CGA-N46. The result showed that CGA-N46 still kept its antifungal to Candida albican after purification.