Perfusion chromatography—a new procedure for very rapid isolation of integral photosynthetic membrane proteins |
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Authors: | Margrit Roobol-Bóza Susana Shochat Staffan E Tjus Åsa Hagman Peter Gast Bertil Andersson |
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Institution: | (1) Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden;(2) Department of Biophysics, Huygens Laboratory, State University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands;(3) Department of Biological Chemistry, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel |
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Abstract: | The biochemical isolation of pure and active proteins or chlorophyll protein complexes has been crucial for elucidating the mechanism of photosynthetic energy conversion. Most of the proteins involved in this process are embedded in the photosynthetic membrane. The isolation of such hydrophobic integral membrane proteins is not trivial, and involves the use of detergents often combined with various time-consuming isolation procedures. We have applied the new procedure of perfusion chromatography for the rapid isolation of photosynthetic membrane proteins. Perfusion chromatography combines a highly reactive surface per bed volume with extremely high elution flow rates. We present an overview of this chromatographic method and show the rapid isolation of reaction centres from plant Photosystems I and II and photosynthetic purple bacteria, as well as the fractionation of the chlorophyll a/b-binding proteins of Photosystem I (LHC I). The isolation times have been drastically reduced compared to earlier approaches. The pronounced reduction in time for separation of photosynthetic complexes is convenient and permits purification of proteins in a more native state, including the maintainance of ligands and the possibility to isolate proteins trapped in intermediate metabolic or structural states.Abbreviations Chl
chlorophyll
- LDAO
N,N dimethyldodecylamine-N-oxide
- LHC
light-harvesting complex
- PS
photosystem
- SDS-PAGE
sodium dodecyl sulphate polyacrylamide gel electrophoresis |
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Keywords: | bacterial reaction centres chlorophyll-binding proteins hydrophobic membrane proteins Photosystem I Photosystem II |
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