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

Djemel Hamdane (1) , Stéphane Skouloubris (2) , Hannu Myllykallio (3) , Béatrice Golinelli-Pimpaneau (1)

1 LEBS - Laboratoire d'Enzymologie et Biochimie Structurales
2 IGM - Institut de génétique et microbiologie [Orsay]
3 LOB - Laboratoire d'optique et biosciences

Folate-dependent tRNA m5U methyltransferase TrmFO is a flavoprotein that catalyzes the C5-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 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. Cop. 2010 Elsevier Inc. All rights reserved.

Mots clés: Expression; Purification; Recombinant TrmFO protein; Flavoenzyme; RNA methyltransferase; 5-Methyluridine; Histidine tag; Expression

Domaines: Biochimie [q-bio.BM]

Djemel Hamdane, Stéphane Skouloubris, Hannu Myllykallio, Béatrice Golinelli-Pimpaneau. Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m5U54 methyltransferase from Bacillus subtilis. Protein Expression and Purification, 2010, 73 (1), pp.83-89. ⟨10.1016/j.pep.2010.04.013⟩. ⟨hal-00805069⟩