Abstract:

The Kringle 5 domain of plasminogen, which was previously shown to inhibit angiogenesis in vitro and in vivo, is one of the most potent angiogenesis inhibitors known to date. A two-step chromatographic method, which consists of Chelating Sepharose Fast Flow chelated with Ni2+ affinity medium and Sephadex G-75 medium, was established to separate and purify recombinant angiogenesis inhibitor Kringle 5 (rK5). Through the two-step chromatographic purification process, the obtained rK5 was confirmed to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and its relative molecular mass was estimated to be about 32000, which matched well with the prediction by gene sequence. Its purity was about 98%, and the total protein recovery of this method was 0.63%. In addition, it was found that it inhibited the blood vessel growth of chick embryo chorioallantoic membrane effectively.

Key words: affinity column chromatography, gel exclusion column chromatography, purification , recombinant angiogenesis inhibitor Kringle 5, separation