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E. COLI EXPRESSION AND PURIFICATION OF HUMAN MITOCHONDRIAL Na+/H+ EXCHANGER (NHE6) A. Kicinska and A. Szewczyk
Recently, mitochondrial Na+/H+ exchanger (NHE6) was cloned [1]. NHE6 plays an important role in maintaining the mitochondrial Ca2+ homeostasis. This is achieved by an integrated cycle of Ca2+ uniport and Na+/H+, and Na+/Ca2+ antiport pathways located in the inner membrane of mitochondria. Until now, the Na+/H+ antiporter was only characterized after partial purification and reconstitution into proteoliposomes. Cloning of NHE6 gives a unique opportunity to study structure-function relationship of the homogenous mitochondrial exchanger in a fully controlled lipid environment. The NHE6 gene was cloned into pET29 expression vector and will be expressed as fusion protein with 6x Histidine residues and S-Tag in Escherichia coli. Expression of NHE6 in E. coli will be achieved under the transcriptional regulation of the T7 promoter. Expressed protein will be purified from inclusion bodies using Ni2+ immobilized on the His-Bind Resin (Novagen). Activity of NHE6 after reconstitution into asolectin proteoliposomes will be studied with pH-sensitive fluorescent probe BCECF (2',7'-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester). [1] Numata et al. (1998) J. Biol. Chem. 273:6951-6959
For further information contact...Carmen Mannella: carmen@wadsworth.org |
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