Expression of Human
Angiostatin in Pichia pastoris and the
Detection of Its Anti-angiogenic Activity
XIN Li, ZHANG
Li1, XU Ren, ZHANG Qian, YE Qin1, LI Zai-Ping, GAN
Ren-Bao*
( Institute of Biochemistry and Cell Biology, Shanghai Institutes for
Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031,
China；1State Key Laboratory of Bioreactor
Engineering and Institute of Biochemistry, East China University of Science and
Technology, Shanghai 200237,
China )
Abstract Human
angiostatin cDNA was amplified from human hepatoma cell line HepG2 using RT-PCR
and was cloned into pPIC9K vector. Recombinant Pichia pastoris strain
with 4 copies of angiostatin gene was obtained. Recombinant protein was
purified by lysine affinity Sepharose column and the finally purified
angiostatin was 25 mg/L, higher than previously reported 17 mg/L. Amino acid
sequence analysis revealed the identity of our protein the same with that previously
reported. Recombinant angiostatin inhibited specifically the proliferation of
bovine aortic endothelial cell stimulated by bFGF, with ED50 being
about 3 mg/L.
Key words angiostatin； Pichia pastoris； multiple copy integration； inhibit； endothelial cell
*Corresponding author： Tel, 86-21-64374430-321； Fax, 86-21-64338357； e-mail, [email protected]