[Expression and purification of the N-domain of human canstatin and its bioactivity]

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2003 May;35(5):483-7.
[Article in Chinese]

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 chicken embryo microcapillary vessel.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiogenesis Inhibitors / biosynthesis*
  • Angiogenesis Inhibitors / isolation & purification
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Gene Expression
  • Humans
  • Placenta / chemistry*
  • Pregnancy Proteins / biosynthesis*
  • Pregnancy Proteins / genetics
  • Pregnancy Proteins / isolation & purification
  • Protein Structure, Tertiary
  • RNA
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • Angiogenesis Inhibitors
  • Pregnancy Proteins
  • Recombinant Proteins
  • RNA