Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m5U54 methyltransferase from Bacillus subtilis

Protein Expr Purif. 2010 Sep;73(1):83-9. doi: 10.1016/j.pep.2010.04.013. Epub 2010 Apr 19.

Abstract

Folate-dependent tRNA m(5)U methyltransferase TrmFO is a flavoprotein that catalyzes the C(5)-methylation of uridine at position 54 in the TPsiC loop of tRNA in several bacteria. Here we report the cloning and optimization of expression in Escherichia coli BL21 (DE3) of untagged, N-terminus, C-terminus (His)(6)-tagged TrmFO from Bacillus subtilis. Tagged and untagged TrmFO were purified to homogeneity by metal affinity or ion exchange and heparin affinity, respectively, followed by size-exclusion chromatography. The tag did not significantly alter the expression level, flavin content, activity and secondary structure of the protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / enzymology*
  • Bacillus subtilis / genetics
  • Bacterial Proteins / biosynthesis*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Chromatography, Affinity / methods
  • Chromatography, Gel / methods
  • Circular Dichroism
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Histidine
  • Methylation
  • Models, Molecular
  • Molecular Weight
  • Mutation
  • NAD / metabolism
  • Oxidation-Reduction
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • tRNA Methyltransferases / biosynthesis*
  • tRNA Methyltransferases / chemistry
  • tRNA Methyltransferases / genetics
  • tRNA Methyltransferases / isolation & purification

Substances

  • Bacterial Proteins
  • Recombinant Fusion Proteins
  • NAD
  • Histidine
  • tRNA Methyltransferases
  • tRNA(uracil-5)-methyltransferase