Expression and purification of the carboxyl terminus domain of Schizosaccharomyces pombe dicer in Escherichia coli

Protein Pept Lett. 2005 May;12(4):311-4. doi: 10.2174/0929866053765590.

Abstract

The carboxyl terminus domain of Schizosaccharomyces pombe dicer (yDicerC) was expressed in Escherichia coli as an MBP-fusion protein (MBP-yDicerC). When the E. coli strain was cultured and induced at 25 degrees C, the MBP-yDicerC was partly expressed in the soluble fraction. It was then purified by two step affinity chromatography with amylose resin and Ni-NTA His Bind(R) resin. The purified MBP-yDicerC showed double-strand RNA digestion activity. siRNA-like products about 22-nt in length were generated.

MeSH terms

  • Carrier Proteins / chemistry
  • Chromatography, Affinity
  • Escherichia coli / genetics*
  • Maltose-Binding Proteins
  • RNA Interference
  • RNA, Double-Stranded / metabolism
  • Recombinant Fusion Proteins / biosynthesis
  • Ribonuclease III / biosynthesis*
  • Ribonuclease III / isolation & purification*
  • Schizosaccharomyces / enzymology*

Substances

  • Carrier Proteins
  • Maltose-Binding Proteins
  • RNA, Double-Stranded
  • Recombinant Fusion Proteins
  • Ribonuclease III