Histidine tagging both allows convenient single-step purification of bovine rhodopsin and exerts ionic strength-dependent effects on its photochemistry

Abstract

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-opshis(6x)). The presence of this tag was confirmed by immunoblot analysis. Incubation with 11-cis- retinal produced a photosensitive pigment (v-Rhohis(6x)) at a level of 15-20 pmol/106 cells. The histidine tag was exploited to purify v-Rhohis(6x) via immobilized metal affinity chromatography. Optimized immobilized metal affinity chromatography yielded a binding capacity of ≥35 nmol of v- Rhohis(6x) per ml of resin and purification factors up to 500. Best samples were at least 85% pure, with an average purity of 70% (A(280 nm)/A(500 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-Rhohis(6x) showed a small but significant red shift (501 ± 1 nm) compared to wild type rhodopsin (498 ± 1 nm). The pK(a) of the Meta I mutually implies Meta II equilibrium in v-Rhohis(6x) 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-Rhohis(6x).