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Expression of mammalian Rab Escort protein-1 and -2 in yeast Saccharomyces cerevisiae

Domenii publicaţii > Biologie + Tipuri publicaţii > Articol în revistã ştiinţificã

Autori: Sidorovitch V, Niculae A, Kan N, Ceacareanu AC, Alexandrov K.

Editorial: Protein Expr Purif, 26(1), p.50-8, 2002.

Rezumat:

Rab GTPases are post-translationally geranylgeranylated on their C-terminal cysteines by Rab geranylgeranyl transferase (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 Saccharomyces cerevisiae 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 scaled up to 30L 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

Cuvinte cheie: REP-1, REP-2, RabGGTase

URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12356470&query_hl=1&itool=pubmed_docsum