Increased peptide deformylase activity for N-formylmethionine processing of proteins overexpressed in Escherichia coli: application to homogeneous rubredoxin production.
Deformylation of the initiator N-formylmethionine does not always proceed to completion for proteins overexpressed in Escherichia coli. To overcome this limitation, the def gene encoding the Escherichia coli peptide deformylase was cloned into the plysS plasmid under the tetracycline (Tc) promoter control. The efficiency of this constitutive level of peptide deformylase expression was demonstrated for the case of the rubredoxins from both mesophilic and hyperthermophilic organisms which normally retain a majority of their N-formyl terminal form. Indicating the potential structural/functional significance of residual formylation, the presence of a highly solvent exposed N-formyl group in rubredoxin is discernable in the amide NMR chemical shifts for the active site metal-coordinating cysteines more than 21A away.
Tang
Jianzhong
J
Wadsworth Center, New York State Department of Health, University at Albany-SUNY, Empire State Plaza, P.O. Box 509, 12201-0509, USA.
Hernández
Griselda
G
LeMaster
David M
DM
D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism*; D019843:Archaeal Proteins / Q000096:biosynthesis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D009239:N-Formylmethionine / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D020027:Pyrococcus furiosus / Q000378:metabolism; D012416:Rubredoxins / Q000096:biosynthesis*; D013752:Tetracycline / Q000494:pharmacology; D015854:Up-Regulation
D019843:Archaeal Proteins; D029968:Escherichia coli Proteins; D012416:Rubredoxins; D009239:N-Formylmethionine; D000581:Amidohydrolases; C084507:peptide deformylase; D013752:Tetracycline