Xu Y, Mayor J A, Gremse D, Wood D O, Kaplan R S
Department of Pharmacology, College of Medicine, University of South Alabama, Mobile 36688.
Biochem Biophys Res Commun. 1995 Feb 15;207(2):783-9. doi: 10.1006/bbrc.1995.1255.
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.
大鼠肝脏线粒体三羧酸转运蛋白已在大肠杆菌中过表达。所表达的转运蛋白含有一个21个氨基酸的N端融合序列，积聚在包涵体中。随后从分离的包涵体中提取三羧酸转运蛋白，每升大肠杆菌培养物可产生约90毫克转运蛋白，纯度大于90%。将纯化的、过表达的转运蛋白整合到磷脂囊泡中后，它催化对1,2,3-苯三羧酸敏感的柠檬酸/柠檬酸交换（即线粒体三羧酸转运蛋白的标志性反应）。重组转运蛋白的动力学特征表明，其Km为0.37 mM，Vmax为101 nmol/min/mg蛋白。重组表达的转运蛋白的底物特异性与天然转运蛋白几乎相同。这些研究代表了大鼠肝脏线粒体三羧酸转运蛋白的首次过表达。通过提供大量高度纯化的、功能正常的转运蛋白，该系统现在将能够进行各种结构研究，包括定点诱变，而这些研究在此之前是无法进行的。
