Inhibition Effect in vitro of Purified Endostatin Expressed in Pichia pastoris
人内皮抑素在毕赤酵母中的表达、纯化与生物功能研究

Endostatin is a newly found inhibitor of angiogenesis, which is identified as c\|terminal 184 amino acid fragment of collagen XVIII NC1\|domain. A 570bp cDNA fragment of endostatin has been amplified by PCR from a commercial human fetal liver cDNA library. After subcloned into the yeast vector pPIC9 and subsequenced to prove its correctness, \%Pichia pastoris\% was transformed with the recombinant pPIC9\|endostatin. The expressed endostatin in \%P.pastoris\% was purified by heparin\|sapherose affinity chromatography. It\'s purity identified by SDS\|PAGE thin layer scanning analysis was up to 98.7% and its Mol. Weight measured by MS was 20.34kD. The expression level was up to 40mg/L. The first fifteen amino acid sequence of the N\|terminal was completely identical with the inner sequence C\|terminal fragment of collagen XVIII NC1 domain as has been designed. Bioassay indicated that the recombinant endostatin can inhibit angiogenesis stimulated by bFGF in CAM test and also the proliferation of both HUVEC and ECV304 in an \%in vitro\% test.