Biomaterial Database

High-yield bacterial expression, purification, and functional reconstitution of the tricarboxylate transport protein from rat liver mitochondria. 1995

Y Xu, and J A Mayor, and D Gremse, and D O Wood, and R S Kaplan

Department of Pharmacology, College of Medicine, University of South Alabama, Mobile 36688.

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.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008081	Liposomes	Artificial, single or multilaminar vesicles (made from lecithins or other lipids) that are used for the delivery of a variety of biological molecules or molecular complexes to cells, for example, drug delivery and gene transfer. They are also used to study membranes and membrane proteins.	Niosomes,Transferosomes,Ultradeformable Liposomes,Liposomes, Ultra-deformable,Liposome,Liposome, Ultra-deformable,Liposome, Ultradeformable,Liposomes, Ultra deformable,Liposomes, Ultradeformable,Niosome,Transferosome,Ultra-deformable Liposome,Ultra-deformable Liposomes,Ultradeformable Liposome
D008930	Mitochondria, Liver	Mitochondria in hepatocytes. As in all mitochondria, there are an outer membrane and an inner membrane, together creating two separate mitochondrial compartments: the internal matrix space and a much narrower intermembrane space. In the liver mitochondrion, an estimated 67% of the total mitochondrial proteins is located in the matrix. (From Alberts et al., Molecular Biology of the Cell, 2d ed, p343-4)	Liver Mitochondria,Liver Mitochondrion,Mitochondrion, Liver
D008970	Molecular Weight	The sum of the weight of all the atoms in a molecule.	Molecular Weights,Weight, Molecular,Weights, Molecular
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002352	Carrier Proteins	Proteins that bind or transport specific substances in the blood, within the cell, or across cell membranes.	Binding Proteins,Carrier Protein,Transport Protein,Transport Proteins,Binding Protein,Protein, Carrier,Proteins, Carrier
D002951	Citrates	Derivatives of CITRIC ACID.
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D004591	Electrophoresis, Polyacrylamide Gel	Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.	Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli