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 Ni2+-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.
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September 1999
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Research Article|
August 24 1999
Large-scale production and purification of functional recombinant bovine rhodopsin with the use of the baculovirus expression system
Corné H. W. KLAASSEN;
Corné H. W. KLAASSEN
1Department of Biochemistry, Institute of Cellular Signalling, University of Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands
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Petra H. M. BOVEE-GEURTS;
Petra H. M. BOVEE-GEURTS
1Department of Biochemistry, Institute of Cellular Signalling, University of Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands
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Godelieve L. J. DECALUWÉ;
Godelieve L. J. DECALUWÉ
1Department of Biochemistry, Institute of Cellular Signalling, University of Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands
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Willem J. DEGRIP
Willem J. DEGRIP
1
1Department of Biochemistry, Institute of Cellular Signalling, University of Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands
1To whom correspondence should be addressed (wdegrip@baserv.uci.kun.nl).
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Publisher: Portland Press Ltd
Received:
March 11 1999
Revision Received:
May 21 1999
Accepted:
June 11 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 342 (2): 293–300.
Article history
Received:
March 11 1999
Revision Received:
May 21 1999
Accepted:
June 11 1999
Citation
Corné H. W. KLAASSEN, Petra H. M. BOVEE-GEURTS, Godelieve L. J. DECALUWÉ, Willem J. DEGRIP; Large-scale production and purification of functional recombinant bovine rhodopsin with the use of the baculovirus expression system. Biochem J 1 September 1999; 342 (2): 293–300. doi: https://doi.org/10.1042/bj3420293
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