Large-scale production and purification of functional recombinant bovine rhodopsin with the use of the baculovirus expression system.
Here we describe a generic procedure for the expression and purification of milligram quantities of functional recombinant eukaryotic integral membrane proteins, exemplified by hexahistidine-tagged bovine rhodopsin. These quantities were obtained with the recombinant baculovirus/Sf9 insect cell-based expression system in large-scale bioreactor cultures with the use of a serum-free and protein-free growth medium. After optimization procedures, expression levels up to 4 mg/l were established. The recombinant rhodopsin could be purified with high overall yield by using immobilized-metal-affinity chromatography on Ni(2+)-agarose. After reconstitution into a native lipid environment, the purified protein was functionally indistinguishable from native rhodopsin with regard to the following parameters: spectral absorbance band, structural changes after photoactivation, and G-protein activation. The procedures developed can be adapted to other membrane proteins. The ability to produce and purify tens of milligrams of functional recombinant eukaryotic membrane protein meets the ever-increasing demand of material necessary to perform detailed biochemical and structural biophysical studies that are essential in unravelling their working mechanism at a molecular level.
Klaassen
C H
CH
Department of Biochemistry, Institute of Cellular Signalling, University of Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands.
Bovee-Geurts
P H
PH
Decaluwé
G L
GL
DeGrip
W J
WJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D019149:Bioreactors; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity; D016895:Culture Media, Serum-Free; D015870:Gene Expression; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012243:Rhodopsin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D018411:Spodoptera
D016895:Culture Media, Serum-Free; D008565:Membrane Proteins; D011994:Recombinant Proteins; D012243:Rhodopsin