Recombinant Vascular Growth Inhibition The Factor Kringle5 Cloning, Expression, Purification And Activity

Angiogenesis is important for tumor growth, its inhibition may be a potential new approach to cancer therapy. The kringle 5 (K5) domain of plasminogen, which was previously shown to inhibit angiogenesis in vitro and vivo, is one of the most potential angiogenesis inhibitors described to date. In this study, the K5 gene was amplified from pET32a-K5 by PCR, and then was inserted into pET15b to construct a prokaryotic vector pET15b-K5. The target protein was highly expressed in E.coli BL21(DE3) and was mainly present in the supernatant of the broken cells observed by SDS-PAGE. The protein was purified by ion exchange chromatography (SP Sepharose Fast-Flow). The result of SDS-PAGE showed that the purity of obtained K5 was about 95%. After desalting with size-exclusion chromatography, it was found that it inhibited the blood vessel growth of chick embryo chorioallantoic membrane effectively.In this paper, the fermentation process of the K5 was studied through the single factor experiments, in order to improve the expression of K5. The experiment was based on the LB medium and the result was measured by the cell dry weight. Through the experiment, the optimal composition of synthetic medium was determined as follows:pancreatic peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, glucose 6 g/L, ammonium chloride 2.6 g/L, disodium hydrogen phosphate 6 g/L, sodium dihydrogen phosphate 5 g/L. In order to determine the optimal induction time, the growth curve was made. On the optimized culture medium, the four factors of the induction dose, time, temperature and rotate speed were analyzed by SDS-PAGE. The optimal culture condition was determined as follows:IPTG 0.01 mM, induction time 6 h, induction temperature 37℃, rotate speed 220 rpm. The total content of K5 was about 20% in the end.The mutational K5 gene whose DNA sequence besides the kringle domain was got rid of was amplified from pET32a-K5 by PCR, and then was inserted into pET25b to construct a prokaryotic vector pET25b-K5.The target protein was highly expressed in E.coli BL21(DE3) and the molecular weight was about 10 KD which was consistent with expectation.