Optimization Of Fermentation For Producing Heat Shock Protein65-MUC1 Fusion Protein

Heat shock protein 65-MUC1(HSP-MUC1) is constructed by fusing BCG HSP65 with MUC1 VNTR peptide gene. It was produced in protokaryon BL21(DE3) strain. In the human system, HSP65-MUC1 loaded human DC induced the generation of autologous MUC1 specific CTL in vitro. These results suggest that exogenously applied HSP65-MUC1 may be used to treat MUC1 tumors by inducing the epitope-specific CTL as well as non-specific anti-tumor response mediated by the HSP65 part of the fusion protein. HSP-MUC1 has been approved by Chinease FDA to conduct Phase I clinical trial in china for thhe treatment of MUC1+ breast-tumor in China(Dapeng Li, 2004).This thesis focuses on optimization of fermentation for producing heat shock protein65-MUC1 fusion protein.1. Screening of strain expressing HSP-MUC1To improve the yield of HSP-MUC1, we have screened the pET28a/HSP-MUC1/BL21(DE3) strain. First, according the expressing level in the test tube, two colones, named No.2 and No.17, were selected. Then, the two strains were cultivated and induced with IPTG in fermentor. Analysising the expression level and yield of the fermentation, we have choosed the No.2 stain to be identified by SDS-PAGE, agarose electrophoresis, Western Blot and sequencing. At last, the strain was lyophilized and stored at 4℃.2. Optimization of culture mediumThe culture medium of fermentation for pET28a/HSP-MUC1/BL21(DE3) was studied in shaking-flask. Firstly, the LB,M9,TB,M9Zb,ZYM media were selected to grow host cell and expression of HSP-MUC1. Comparing the expression level and cell density, we concluded that M9ZB is the best culture medium. Secondly, using a single factor experiment, the substances of C and N were determined. The results showed that glycerol is the best carbon source, and casin is the best organic nitrogen sources. At this time the culture medium was named M9-1. Thirdly, 3 steps of orthogonal tests was carried out, the concentration of carbon, nitrogen, mineral salt and trace element were determined according the orthogonal tests results. At last the culture medium was named M9-3.3. Optimization of the fermentationThe expression level of HSP-MUC1 in shaking-flask was more than 50% of the total bacteria extracts on the conditions (37℃, pH 7.0, 5% inoculum, 0.25mM IPTG and 3-4 hours of induction timing). According to this results, the conditions of fermentation was determined.4.Selection of feed concentration and Fed-batch modelFeeding of three C:N ratio was added when the substances were exhausted. It was showed that C:N=2:1 was the best ratio.Three Fed-batch strategies (constant, gradient and DO2-stat) were applied to observe the expression and yield of HSP-MUC1. It was found that the DO2-stat fed-batch model was the best. Above all , utilizing the screened strain , M9-3 medium, optimum culture conditions and DO2-stat fed-batch model, the final expression level of HSP-MUC1 is about 59%, the yield is 5.4g/L(DCW).