[Inhibition effect in vitro of purified endostatin expressed in Pichia pastoris].
Endostatin is a newly found inhibitor of angiogenesis, which is identified as c-terminal 184 amino acid fragment of collagen XVIII NC1-domain. A 570 bp 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 subsequence 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 20.34 kD. The expression level was up to 40 mg/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.
Feng
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Beijing Institute of Biotechnology, Beijing, 100850, China. feifeicat@mailcity.com
Cui
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LB
Liu
C X
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Ma
Q J
QJ
D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D002642:Chick Embryo; D003094:Collagen / Q000494:pharmacology*; D043170:Collagen Type XVIII; D043169:Endostatins; D016222:Fibroblast Growth Factor 2 / Q000037:antagonists & inhibitors; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D010843:Pichia / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000494:pharmacology
D020533:Angiogenesis Inhibitors; D043170:Collagen Type XVIII; D043169:Endostatins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D003094:Collagen