High Expression Of Recombinant [Gly～(14)]-Humanin And Human Insulin Mutant In Escherichia Coli And Purification

Objective1 . Construction of prokaryotic high expression vector of [Gly14]-Humanin (HNG) and its expression and purification.2. Construction of prokaryotic high expression vector of human insulin mutant (M-insulin) and its expression, purification and bioassay.MethodsBy gene synthesis, we get the cDNAs of [Gly14]-Humanin and human insulin mutant, and then we construct three prokaryotic expression vectors: pBV220-HNG, pTXB1-HNG and pTXB1 - M-insulin by DNA polymerase chain reaction(PCR) and DMA recombinant technics. The expression plasmids are identified with PCR and DNA sequencing.pBV220-HNG is transformed into E. coli DH5 a , BL21(DE3) and induced by increasing the temperature to 42℃. pTXB1-HNG and pTXB1-M-insulin are transformed into BL21(DE3) and induced by adding IPTG. We identified the expressed proteins by SDS-PAGE and Western-blot. After the expression form analysis, the insoluble recombinant proteins was purified by destraction and abstersion of inclusion bodies. To study the abstersion condition of the inclusion bodies, we adopted ultrasound crushing and freezing-melting methods. Radioimmunoassay was used to test the immune activity of M-insulin fusion protein.Results1. DNA sequencing demonstrated that we have constructed the expression plasmids pBV220-HNG, pTXB1-HNG and pTXB1-M-insulin successfully. Expression of pBV220-HNG can not be detected in E.coli. pTXB1-HNG and pTXB1-M-insulin are expressed in E.coli successfully. After SDS-PAGE and densitometric scan analysis, the results show that the expression level of HNG fusion protein is above 40% and M-insulin fusion protein above 50% of total bacterial proteins. Western-blot result demonstrated M-insulin fusion protein had specific reaction with mouse anti human insulin antibody(IgG).2. The results show that the optimal conditions of the expression of BL21(DE3)- pTXBl -HNG and BL21(DE3)-pTXB1-M-insuIin are: BL Medium, 37℃, 200rpm for 4.75h, then 1PTG was added to the medium to 0.01mmol/L , induce for another 5h.3. HNG fusion protein and M-insulin fusion protein are expressed as inclusion bodies in E.coli. After washing with reagent (50mmol/L pH8.0 Tris-HCl, 100mmol/L NaCl, 0.5mmol/L EDTA, 2mol/L carbamide, 0.2% Triton X-100, 0.2% DOC ) , ultrasound crushing (200 W, 150 times, 3 seconds per time, spacing3 seconds) and freezing-melting methods, we got HNG fusion protein and M-insulin fusion protein with purity of above 80%.4. Radioimmunoassay result shows that the radioimmune activity of of M-insulin fusion protein is 0.5 unit per litre bacterial liquid.ConclusionWe have constructed the expression plasmids pTXBl-HNG and pTXBl -M-insulin successfully. After SDS-PAGE and densitometric scan analysis, the expression level of HNG fusion protein is above 40% and M-insulin fusion protein above 50%. Western-blot result demonstrated M-insulin fusion protein had specific reaction with mouse anti human insulin antibody, we got HNG fusion protein and M-insulin fusion protein with purity of above 80%. Radioimmunoassaygets the radioimmune activity of of M-insulin fusion protein is 0.5 unit per litre bacterial liquid. Our study establish a stable base for further reseach of HNG and human insulin mutant and provide a theoretics gist for expression of low molecular weight proteins in prokaryotic cells.