Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m5U54 methyltransferase from Bacillus subtilis.
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.
Hamdane
Djemel
D
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 1 Avenue de la Terrasse, 91190 Gif-sur-Yvette, France.
Skouloubris
Stéphane
S
Myllykallio
Hannu
H
Golinelli-Pimpaneau
Béatrice
B
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006639:Histidine; D008745:Methylation; D008958:Models, Molecular; D008970:Molecular Weight; D009154:Mutation; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012359:tRNA Methyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D009243:NAD; D006639:Histidine; D012359:tRNA Methyltransferases; C022769:tRNA(uracil-5)-methyltransferase