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- Title
A simple protocol for the isolation of proteorhodopsins of the dinoflagellate Oxyrrhis marina.
- Authors
Rhiel, Erhard; Nguyen, Tien; Wöhlbrand, Lars; Rabus, Ralf
- Abstract
For the first time, native proteorhodopsins of the marine dinoflagellate Oxyrrhis marina were isolated. Total cell membrane fractions were minced in a bead beater and solubilized with the detergent Triton X‐100. Subsequent sucrose density gradient centrifugation resulted in three or four red‐colored bands. Nonsolubilized, but still red colored, membranes sedimented at the bottom. For each of these bands, absorbance maxima were registered at approximately 514–516 nm with shoulders toward shorter wavelengths (470–490 nm). Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed that the uppermost band represented free retinal chromophore, as it contained no protein. The other bands were almost pure proteorhodopsin fractions as the banding patterns showed one major protein of 25 kDa. Tryptic, in‐gel digestion of the 25 kDa proteins and of faint protein bands above and below 25 kDa was followed by mass spectrometry, confirming these protein bands to consist, nearly exclusively, proteorhodopsins. Only single peptides of few other proteins were detected. In total, at least seven predicted proteorhodopsin protein sequences were experimentally verified.
- Subjects
POLYACRYLAMIDE gel electrophoresis; DENSITY gradient centrifugation; TRITON X-100; AMINO acid sequence; GEL electrophoresis
- Publication
Journal of Basic Microbiology, 2020, Vol 60, Issue 4, p351
- ISSN
0233-111X
- Publication type
Article
- DOI
10.1002/jobm.201900594