Optimization Of Fermentation Process Conditions And Purification For Recombination E.Coli Expressing Angiogenesis Inhibitor Kringle5

Optimization of fermentation process conditions and purification for recombination E.Coli expressing tumor angiogenesis inhibitor kringle5, improving the expressing quatity of kringle5 and search for a simple, rapid and effective purification process is the aim of this paper. In this paper, the optimization of fermentation process and purification of kringle5 in prokaryotic system were studied. On the basis of constructing of a novel recombination E.Coli BL21(DE3)/ pET32a-K5 expressing kringle5, the optimal fermentation culture medium was determined by adjusting the category and composition of nitrogen source and inorganic salt and the ratio of carbon source and nitrogen source in fermentation solutions. The composition of the optimal fermentation culture medium (gramme per liter) is yeast extract 10, ammonium chloride 1.33, sodium dihydrogen phosphate 2, disodium hydrogen phosphate 5, magnesium sulfate with seven water molecule 1 and glucose 6. The optimization of batch fermentation was studied by orthogonal experiment in triangular flask and the optimized fermentation condition is: temperature 35℃, pH 7.0, the amount of inoculation 8%, agitation 260 r/min, the concentration of inducer 0.8 mmol/L, induced phase intermediate stage of the growth, and induced time 6h. And by batch cultivation in fermenter, the optimal dissolved oxygen in fermentation solution was found to be 40%. Based on the above optimal processing variables, the expression of kringle5 can reach 0.59 g/L. And finally, the kringle5 was purified on Cu2+ chelated on Sepharose Fast Flow and CM Sepharose CL 6B cation-exchange medium and its purity is about 96%. The yield of purified kringle5 is 0.3% and the obtained kringle5 has the activity of inhibiting the growth of vascular from chick chorioallantoic membrane.