Functional Expression of His-Tagged Rhodopsin in Sf9 Insect Cells

Abstract

The structure/function relationship of G protein-coupled receptors constitutes one of the most challenging areas of the present postgenomic era. It is estimated that at least a quarter of all human proteins are membrane proteins, with most of them belonging to the superfamily of GPCRs. Not only the relative number of GPCRs, but especially the key roles they play in cell function—and thus their pharmacological importance—warrant the attention they are presently receiving as a major focus of research. Indeed, more than 50% of the presently marketed medication targets this GPCR superfamily. This confronts us with the fact that their activation mechanism is still poorly understood, owing to a serious lack of information on structure and structure/function relationships. For almost all GPCRs, only models are available for the prediction of affinity, specificity, and/or effect of putative (ant)agonists. Detailed information on structure and function is not only hampered by the fact that, being integral membrane proteins, GPCRs are notoriously difficult to purify, but also by the lack of natural sources in which GPCRs are abundantly present. The rod visual pigment rhodopsin, a prototype of a major subclass of the GPCRs, is the only GPCR that can be isolated from native tissue in sufficient amounts to allow biochemical and biophysical studies. However, for detailed studies on the complex structure/function relation at the molecular level, there is a need for functional expression of recombinant protein (see Note 1).