Folate-dependent tRNA m(5)U methyltransferase TrmFO is a flavoprotein that catalyzes the C-5-methylation of uridine at position 54 in the T Psi C loop of tRNA in several bacteria. Here we report the cloning and optimization of expression in Escherichia colt 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. (C) 2010 Elsevier Inc. All rights reserved.