Purification of branched-chain keto acid dehydrogenase regulator from Pseudomonas putida.
BkdR can be isolated in nearly pure form as a tetramer by this procedure, which involves hyperexpressing bkdR from a plasmid, purification by chromatography on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, and dialysis to precipitate BkdR. BkdR is relatively insoluble in aqueous buffers but can be kept in solution in buffer with 50% (v/v) glycerol and 0.2 M NaCl. Cultures of E. coli DH5 alpha (pJRS119) should be maintained at 30 degrees to promote plasmid stability. Because BkdR is prone to form intermolecular disulfide bonds, buffers for SDS-PAGE should contain fresh 0.5% (v/v) 2-mercaptoethanol.
Madhusudhan
K T
KT
Central Arkansas Veterans Healthcare System, Pathology and Laboratory Medicine, Little Rock 72205, USA.
Sokatch
J R
JR
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007658:Ketone Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*
D009097:Multienzyme Complexes; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)