Purification of ribulose-1, 5-bisphosphate carboxylase/oxygenase with high specific activity by fast protein liquid chromatography |
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| Authors: | M E Salvucci A R Portis W L Ogren |
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| Institution: | 1. CNRS, Centre de Recherche de Gif – FRC3115, Institut de Neurobiologie Alfred Fessard – FRC2118, Laboratoire de Neurobiologie et Développement – UPR3294, F-91198 Gif-sur-Yvette, France;2. University of Nha Trang, Institute of Biotechnology and Environment, Nha Trang, Khanh Hoa 57000, Viet Nam;3. CNRS, Centre de Recherche de Gif – FRC3115, Institut de Chimie des Substances Naturelles – UPR 2301, F-91198 Gif-sur-Yvette, France;4. CEA, iBiTec-S, Service d''Ingénierie Moléculaire des Protéines, F-91191 Gif-sur-Yvette, France;1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK;1. Department of Plant Physiology and Anatomy, Institute of Experimental Biology, Faculty of Science, Masaryk University Brno, Kotlářská 2, 611 37, Brno, Czech Republic;2. Department of Physiology, Faculty of Medicine, Masaryk University Brno, Kamenice 753/5, 625 00, Brno, Czech Republic |
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| Abstract: | A rapid procedure for the purification of ribulose-1, 5-bisphosphate carboxylase/oxygenase (rubisco) (EC 4.1.1.39) by fast protein liquid chromatography (FPLC) is described. Chloroplasts isolated mechanically from spinach leaves were used as the source of a stromal extract enriched in rubisco. By subsequent fractionation of this extract on ion-exchange FPLC, highly purified rubisco (sp act 2.10-2.76 mumol/mg protein X min) was obtained in less than 30 min. The high specific activity and excellent stability of the final preparation can be attributed to the use of chloroplasts as a starting material and the short time required for the chromatographic separation, both of which minimize proteolytic activity. |
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