Expression and characterization of bovine mitochondrial methionyl-tRNA transformylase.
Translational initiation in bacteria and some organelles such as mitochondria and chloroplasts requires formyl-methionyl-tRNA (fMet-tRNA). Methionyl-tRNA (Met-tRNA) undergoes formylation by methionyl-tRNA transformylase (MTF), and the resulting fMet-tRNA is utilized exclusively in the initiation process. The gene encoding mammalian mitochondrial MTF (MTFmt) was cloned recently. When the cDNA corresponding to mature MTFmt was cloned into an expression vector, no expression of MTFmt was observed. However, if the cDNA was fused with the histidine-tag sequence at the N-terminus, MTFmt could be expressed in Escherichia coli. The recombinant enzyme was purified by a single step on a histidine-binding metal affinity column. We previously found that native MTFmt is able to formylate E. coli elongator Met-tRNA as well as the initiator Met-tRNA. The specific formylation of the initiator Met-tRNA by E. coli MTF is quite important in bacterial translational initiation. The purified recombinant MTFmt with the histidine-tag showed almost identical kinetic parameters to those of native MTFmt. This expression system is suitable for the rapid, efficient production of MTFmt in amounts adequate for further biophysical studies, which will provide another approach for elucidating the formylation mechanism, in addition to studies on E. coli MTF.
Takeuchi
N
N
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
Ueda
T
T
Watanabe
K
K
D000818:Animals; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics; D019877:Hydroxymethyl and Formyl Transferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D019877:Hydroxymethyl and Formyl Transferases; C021561:methionyl-tRNA formyltransferase