[Expression and purification of the N-domain of human canstatin and its bioactivity].
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 chicken embryo microcapillary vessel.
He
Guo-An
GA
Key Laboratory of Genetic Engineering of Ministry of Education, Department of Biochemistry, Zhongshan University, Guangzhou 510275, China.
Luo
Jin-Xian
JX
Zhang
Tian-Yuan
TY
Hu
Zhi-Shang
ZS
Gu
Qu-Liang
QL
D020533:Angiogenesis Inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D010920:Placenta / Q000737:chemistry*; D011257:Pregnancy Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D012313:RNA; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020533:Angiogenesis Inhibitors; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; D012313:RNA