| Autor: |
Janssen, Jacques J.M., Bovee-Geurts, Petra H.M., Merkx, Maarten, DeGrip, Willem J. |
| Zdroj: |
Journal of Biological Chemistry; May 1995, Vol. 270 Issue: 19 p11222-11229, 8p |
| Abstrakt: |
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 frugiperdacells 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/106cells. The histidine tag was exploited to purify v-Rhohis6xvia immobilized metal affinity chromatography. Optimized immobilized metal affinity chromatography yielded a binding capacity of ≥35 nmol of v-Rhohis6xper ml of resin and purification factors up to 500. Best samples were at least 85% pure, with an average purity of 70% (A280nm/α500nm= 2.5 ± 0.4, n= 7). Remaining contamination was largely removed upon reconstitution into lipids, yielding rhodopsin proteoliposomes with a purity over 95%. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
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