Histidine tagging both allows convenient single-step purification of bovine rhodopsin and exerts ionic strength-dependent effects on its photochemistry.
For rapid single-step purification of recombinant rhodopsin, a baculovirus expression vector was constructed containing the bovine opsin coding sequence extended at the 3'-end by a short sequence encoding six histidine residues. Recombinant baculovirus-infected Spodoptera frugiperda cells produce bovine opsin carrying a C-terminal histidine tag (v-opshis6x). The presence of this tag was confirmed by immunoblot analysis. Incubation with 11-cis-retinal produced a photosensitive pigment (v-Rhohis6x) at a level of 15-20 pmol/10(6) cells. The histidine tag was exploited to purify v-Rhohis6x via immobilized metal affinity chromatography. Optimized immobilized metal affinity chromatography yielded a binding capacity of > or = 35 nmol of v-Rhohis6x per ml of resin and purification factors up to 500. Best samples were at least 85% pure, with an average purity of 70% (A280 nm/A500 nm = 2.5 +/- 0.4, n = 7). Remaining contamination was largely removed upon reconstitution into lipids, yielding rhodopsin proteoliposomes with a purity over 95%. Spectral analysis of v-Rhohis6x showed a small but significant red shift (501 +/- 1 nm) compared to wild type rhodopsin (498 +/- 1 nm). The pK alpha of the Meta I<==>Meta II equilibrium in v-Rhohis6x is down-shifted from 7.3 to 6.4 resulting in a significant shift at pH 6.5 toward the Meta I photointermediate. Both effects are reversed upon increasing the ionic strength. FT-IR analysis of the Rho-->Meta II transition shows that the corresponding structural changes are identical in wild type and v-Rhohis6x.[1]References
- Histidine tagging both allows convenient single-step purification of bovine rhodopsin and exerts ionic strength-dependent effects on its photochemistry. Janssen, J.J., Bovee-Geurts, P.H., Merkx, M., DeGrip, W.J. J. Biol. Chem. (1995) [Pubmed]
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