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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