| Recently, it had been got much focus on producing special functional proteins by biotechnology methods. Thus it was much more important to improve the expression level of the valuable proteins such as medical proteins. High-density fermentation (HDF) is one of the important way to improve the growth of recombinant strain, so many scientists prefer to use this method to increase the expression level of heroge-neous proteins. Fed-batch fermentation (FBF) is the useful technology to increase the yield of recombinant strain in HDF. The purpose of this study reported here is to improve the expression levels of Cecropin-XJ one of the antimicrobial peptides, using the HDF approach and to purify this protein as well as test its biological function .In the experiment of shake-flask fermentation (SFF), the conditions for high-density culture of a genetic engineering strain (GES) expressing Cecropin-XJ were studied, including the effect of various carbon sources, different amounts of yeast powder, different concentrations of peptone and KH2PO4, and initial pH values on the GES .Then the orthogonal design experiment was employed to analyze the mutual effects between these factors so that the optimal media were obtained .These results showed that the composition of the optimized media was 3% yeast powder, 2% peptone, 4% glycerol , and the initial pH value was 6. In this experiment the optical density (OD600) of the GES reached 48.92±0.59 in the optimum conditions three times higher than that in the original culture conditions ,and meanwhile the antimicrobial activity of supernatant containing Cecropin-XJ increased considerably. As for HDF techniques, firstly the effects of age and amounts of bacterial seeds on HDF were studied by SFF. Then the GES were investigated by SFF to determine the parameters for FBF to achieve an applicable process of FBF. The optimized factors we got are as following:(1) the age of bacterial seeds was 12~24h after been activated and the amounts of the seeds was 2%~8% of total volumes. (2) the optimal feeding time was at the middle-end of the logarithm-growth phase.(3)the optimal feeding substrate was glycerol.(4)the optimal feeding amounts was 40mg/ml. Based on the above experimental results the high-density fermentation was conducted with the NBS-5L fermentor with multi-sensors .In the fermentation cells were growing for 8h,and the feeding operation using glycerol as carbon source was lasted from 8h to 24h,and then the fermentation ended at 48h.In the whole process ,the growth of cells was monitored by measuring OD600 of samples keeping the dissolved oxygen above 20%.The results showed that the OD600 of the cells could reach 64 and the maximum expression level of total protein was 0.6mg/ml fifty percent higher than that in the SFF. Subsequently, the preliminary purification of recombinant Cecropin-XJ expressed in supernatent was carried out after the primary characterization of the protein. According to Agarose Diffusion Assay, this recombinant Cecropin-XJ exhibited an extreme heat-stable property and strong antibacterial effect .The test of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) showed that MIC of the supernatant against Gram-positive bacteria was 3.25mg/ml. The main inhibit effect of the protein on bacteria appeared probably by killing the cells. After detecting the antibacterial activity and characters of Cecropin-XJ, salting out and gel chromatography with sephadex G-75 were used to purify the expressed protein from the supernatant. After the gel chromatography, a single protein peak in the separation graph was observed with obvious antibacterial activity. SDS-PAGE analysis confirmed that the Cecropin-XJ was purified primarily.
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