Rhodopsin kinase: Expression in mammalian cells and a two-step purification
Open Access
- 28 March 2000
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 97 (7) , 3004-3009
- https://doi.org/10.1073/pnas.97.7.3004
Abstract
A suitable system for expression of the rhodopsin kinase (RK) gene and its mutants is needed for structure–function studies of RK. Previously, investigation of the baculovirus system showed satisfactory production of RK, but posttranslational isoprenylation was deficient. We now report on a comparative study of expression of the RK gene in yeast (Pichia pastoris), COS-1 cells and in an HEK293 stable cell line. Expression in COS-1 cells, by using pCMV5 vector, is the most satisfactory. A two-step procedure for purification of the expressed enzyme with an N-terminal histidine tag has been developed. The purified enzyme has correct posttranslational modifications and shows a somewhat broader pH vs. catalytic activity profile than the wild-type enzyme.Keywords
This publication has 19 references indexed in Scilit:
- G PROTEIN–COUPLED RECEPTOR KINASESAnnual Review of Biochemistry, 1998
- Structure and function in rhodopsin: Peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinaseProceedings of the National Academy of Sciences, 1997
- Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines.Proceedings of the National Academy of Sciences, 1996
- Purification of Rhodopsin Kinase from Bovine Rod Outer SegmentsPublished by Elsevier ,1993
- Isoprenylation in regulation of signal transduction by G-protein-coupled receptor kinasesNature, 1992
- [2] Protein kinase catalytic domain sequence database: Identification of conserved features of primary structure and classification of family membersPublished by Elsevier ,1991
- Expression of a synthetic bovine rhodopsin gene in monkey kidney cells.Proceedings of the National Academy of Sciences, 1987
- [35] Preparation of antibodies to rhodopsin and the large protein of rod outer segmentsPublished by Elsevier ,1982
- A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye BindingAnalytical Biochemistry, 1976
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976