de.mpg.escidoc.pubman.appbase.FacesBean
English
 
Help Guide Disclaimer Contact us Login
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT

Released

Journal Article

Expression of mammalian Rab Escort Protein-1 and -2 in yeast <i>Saccharomyces cerevisiae</i>

MPS-Authors

Sidorovitch,  Vadim
Max Planck Institute of Molecular Physiology, Max Planck Society;

Niculae,  Anca
Max Planck Institute of Molecular Physiology, Max Planck Society;

Ceacareanu,  Alice-Corina
Max Planck Institute of Molecular Physiology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons98674

Alexandrov,  Kirill
Abt. III: Physikalische Biochemie, Max Planck Institute of Molecular Physiology, Max Planck Society;

Locator
There are no locators available
Fulltext (public)
There are no public fulltexts available
Supplementary Material (public)
There is no public supplementary material available
Citation

Sidorovitch, V., Niculae, A., Kan, N., Ceacareanu, A.-C., & Alexandrov, K. (2002). Expression of mammalian Rab Escort Protein-1 and -2 in yeast <i>Saccharomyces cerevisiae</i>. Protein Expression and Purification, 26(1): 1, pp. 50-58. Retrieved from http://dx.doi.org/10.1016/S1046-5928(02)00506-5.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0014-0F40-3
Abstract
Rab GTPases are post-translationally geranylgeranylated on their C-terminal cysteines by Rab geranylgeranyl translferase (RabGGTase) and this modification is essential for their biological activity. Rab Escort Protein (REP) is a molecular chaperone that assists in the prenylation reaction carried out by RabGGTase. Mutations in the REP-1 gene lead to progressive retinal degradation and blindness in humans. Despite the significant interest in REP proteins, their preparative production remains challenging. We report here an inducible expression system for the production of REP-1 and REP-2 in yeast Saccharomtyees eerecisiae at levels 3.5 and 2mg/L, respectively. The REP-1 was found to be toxic for yeast cells and its toxicity is proposed to be associated with the formation of unproductive Ypt:REP-1 complexes. To minimize the toxic effect of REP-1 the recombinant protein expression was induced at the end of the exponential phase. Under these conditions, the GAL1 promoter is no longer repressed due to exhaustion of glucose and utilization of ethanol as a carbon source. This expression procedure was successfully sealed up to 30 L for both proteins. The REP-1 and REP-2 were purified using a combination of affinity and anion-exchange chromatography. Purified proteins were functionally active, as determined by a fluorescent Rab binding assay and in vitro prenylation. The reported procedure provides a reliable source of REP proteins for biochemical and structural studies. (C) 2002 Elsevier Science (USA), All rights reserved.