The Separation And Purification Of Recombinant Angiogenesis Inhibitor Kringle 5

The kringle 5 domain of plasminogen, which was previously shown to inhibit angiogenesis in vitro and vivo, is one of the most potent angiogenesis inhibitors described to date.In order to separate and purify recombinant angiogenesis kringle 5, many different kinds of methods were studied. At last, a two-step chromatographic method, which consists of a Chelating Sepharose Fast Flow medium chelated with Ni²⁺ and a Sephadex G-75 medium, was established. The affinity chromatographic column of Chelating Sepharose Fast Flow chelated with Ni²⁺ was 20×1.0cm i.d., Buffer A was consisted of 20mmol/L phosphate buffered saline (PBS, pH7.4), and Buffer B was consisted of 20mmol/L PBS (pH7.4) and 0.5mol/L imidazole. Taken the crude protein solution as the sample and the sample size was 15 mL. Elute with Buffer A after sampled until the column reached equilibrium. Repeat to wash using Buffer A and Buffer B containing increasing percent of Buffer B (i.e. 10%, 50% and 100%). The flow rate of elution solution was 2.0 mL/min. The Sephadex G-75 column was 100×1.5 cm i.d. and eluted with 10mmol/L PBS. The sample size was 8ml and the flow rate of elution solution was 1.0 mL/min. Through this two-step chromatographic purification process, the obtained protein was proved to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and its relative molecular weight was estimated to be about 32 000, which was corresponded with prediction by gene sequence. Its purity was about 96%, and the total protein recovery of this method was 0.63%. Meanwhile, it can inhibit the blood vessel growth of chick embryo chorioallantoic membrane (CAM) effectively.