A simple protocol for the isolation of proteorhodopsins of the dinoflagellate Oxyrrhis marina

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.

The proteorhodopsins of the dinoflagellate Oxyrrhis marina: ultrastructure and localization by immunofluorescence light microscopy and immunoelectron microscopy

“…The pooled proteorhodopsins present in the sucrose density gradient bands B2, B3, and B4 showed a protein with a molecular weight of 25 kDa when subjected to SDS-PAGE. This result is in line with already published molecular weights determined for proteorhodopsins (Rhiel et al 2020 ; Slamovits et al 2011 ) and fits to the predicted values of proteorhodopsins of O. marina . Rhiel et al ( 2020 ) registered a diffuse protein band immediately above and additional faint bands above and below the 25 kDA protein band.…”

“…Fractions, i.e., bands of native proteorhodopsins of Oxyrrhis , were obtained by sucrose density gradient centrifugation as described by Rhiel et al ( 2020 ). The bands were withdrawn from the centrifuge tubes with either a syringe or a pipette and concentrated by diluting them with distilled water, followed by ultracentrifugation for 20 h at 8 °C, using a 70 Ti-fixed angle rotor and a L8-55M ultracentrifuge (Beckman Coulter, Krefeld, Germany).…”

“…The sucrose bands (i.e., bands B2, B3, and B4) from several experiments were pooled, diluted with distilled water, and concentrated by centrifugation as described by Rhiel et al ( 2020 ). The resulting pelleted protein fraction, showing only one protein of 25 kDa was used for immunization.…”

“…They were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. Subsequent mass spectrometry (MS) confirmed that in total at least 7 proteorhodopsins were present in three of the four sucrose bands, i.e., bands B2, B3, and B4 (Rhiel et al 2020 ).…”

“… At least 7 proteorhodopsin sequences of Oxyrrhis marina were recently proven in bands obtained by sucrose density gradient centrifugation, and MS analyses revealed that the bands consisted almost of pure, native proteorhodopsins (Rhiel et al 2020 ). The proteorhodopsin fractions, i.e., bands B2, B3, and B4 were subjected to transmission electron microscopy.…”