Molecular Cloning and Expression of the cDNA Encoding Angiogenesis Inhibitor K4K5 with Pichia pastoris
血管生成抑制因子K4K5 Cdna基因的克隆及其在毕赤酵母中的表达

Kringles of human plasminogen except kringle 4 can inhibit the endothelial cell growth. To determine whether recombinant plasminogen kringle 4-5 (r-K4K5) can inhibit the growth of bovine capillary endothelial (BCE) cell and the angiogenesis of chick embryo chorioallantoic membrane (CAM), we cloned and constructed the modified cDNA region encoding kringles 4 and 5 (K4K5) of human plasmin (ogen) and expressed it with pichia multi-copy expression system. One clone, with the most productive expression was selected from hundreds of transformants. Our data showed that the expression product r-K4K5 (MW 21.5 kD) amounted to 150-250 mg/liter, over 80% of the total secreted protein. It could, dose-dependently, inhibit BCE cell proliferation and inhibit chick embryo CAM angiogenesis.