Purification and folding of recombinant bovine oxoglutarate/malate carrier by immobilized metal-ion affinity chromatography.
A major obstacle to investigating the structure of membrane proteins is the difficulty in obtaining sufficient amounts of functional protein. The oxoglutarate carrier, an intrinsic membrane-transport protein of the inner membranes of bovine-heart mitochondria, has been cloned as a fusion protein containing a C-terminal hexa-histidine tag. This fusion protein has been expressed at an abundant level in a mutant strain of Escherichia coli BL21 (DE3) called C41 (DE3). The protein accumulated as inclusion bodies and none was detected in the bacterial inner membrane. The denatured protein was refolded to reconstitute functional properties similar to the native protein. Solubilized inclusion body protein was immobilized using nickel-chelating affinity chromatography, and purified and refolded in a single step. The protein eluted as a monomer which was stable in mild detergent, at a yield equivalent to 15 mg active protein/liter bacterial culture. The reconstituted fusion protein displayed the same transport characteristics as the wild-type, demonstrating that the tag does not perturb the structure of the protein. The oxoglutarate carrier is one member of an extensive family of mitochondrial transport proteins. These proteins transport many different metabolites across the inner mitochondrial membrane and share a common mechanism of action. Therefore, it is likely that this folding protocol can be applied successfully to other mitochondrial transport proteins, thus providing sufficient protein for extensive crystallization trials with a wide range of family members.
Smith
Vernon R
VR
Medical Research Council, Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, UK.
Walker
John E
JE
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008929:Mitochondria, Heart / Q000378:metabolism; D024101:Mitochondrial Proteins; D009532:Nickel / Q000737:chemistry*; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D002352:Carrier Proteins; D007656:Ketoglutaric Acids; D008081:Liposomes; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; C066909:2-oxoglutarate-malate carrier protein; D009532:Nickel