Expression
and Purification of the N-domain of
Human Canstatin and Its Bioactivity
HE Guo-An,
LUO Jin-Xian*, ZHANG Tian-Yuan, HU Zhi-Shang, GU Qu-Liang
( Key Laboratory of Genetic Engineering of Ministry of
Education, Department of Biochemistry, Zhongshan University, Guangzhou 510275,
China )
Abstract Total RNA was extracted from placenta
umbilical tissue. The canstatin cDNA was amplified from total RNA by net-RT-PCR
technique and cloned into pSP72, and the resulted plasmid pSP72C was used as
template to amplify its N-domain. The amplified 1–89 aa N-domain was then cloned
into pET-3c. The resulted plasmid pET-CN was transformed into E. coli
BL21(DE3). The N-domain was efficiently expressed after IPTG induction as a 10 kD
band on SDS-PAGE. The expressed product accounted for approximately 35.3 % of
the total bacterial proteins, as estimated by densitometry and existed mainly
as inclusion body. The inclusion bodies were washed, lysed and the reactivated
proteins were purified by the Sephadex G-100 gel filtration to a purity of 92.6%.
CAM assay showed that N-domain effectively inhibited the angiogenesis of
chichen embryo microcapillary vessel.
Key words N-domain; gene expression;
protein purification; anti-angiogenesis
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