High-yield bacterial expression, purification, and functional reconstitution of the tricarboxylate transport protein from rat liver mitochondria.
The rat liver mitochondrial tricarboxylate transport protein has been overexpressed in E. coli. The expressed transporter, which contains a 21 amino acid N-terminal fusion sequence, accumulates in inclusion bodies. Subsequent extraction of the tricarboxylate transporter from isolated inclusion bodies yields approximately 90 mg of transport protein per liter of E. coli culture at a purity of greater than 90%. Upon incorporation into phospholipid vesicles the purified, overexpressed transporter catalyzes a 1,2,3-benezenetricarboxylate-sensitive citrate/citrate exchange (i.e., the defining reaction of the mitochondrial tricarboxylate transporter). Kinetic characterization of the reconstituted transporter indicates a Km of 0.37 mM and a Vmax of 101 nmol/min/mg protein. The substrate specificity of the reconstituted, expressed transporter is virtually identical to that of the native transporter. These studies represent the first overexpression of the rat liver mitochondrial tricarboxylate transporter. By providing a large amount of highly-purified, functionally competent transporter this system will now enable a variety of structural studies, including site-directed mutagenesis, which heretofore could not be performed.
Xu
Y
Y
Department of Pharmacology, College of Medicine, University of South Alabama, Mobile 36688.
Mayor
J A
JA
Gremse
D
D
Wood
D O
DO
Kaplan
R S
RS
D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D002951:Citrates / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D008081:Liposomes; D008930:Mitochondria, Liver / Q000378:metabolism*; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
D002352:Carrier Proteins; D002951:Citrates; D017931:DNA Primers; D008081:Liposomes; D011994:Recombinant Proteins; C020723:citrate-binding transport protein