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Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli
Published on: August 3, 2017
Functional relationships between the Saccharomyces cerevisiae cis-prenyltransferases required for dolichol
Kariona Grabińska1, Grazyna Sosińska, Jacek Orłowski
1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
Abstract:
In the yeast Saccharomyces cerevisiae the RER2 and SRT1 genes encode Rer2 and Srt1 proteins with cis-prenyltransferase (cis-PT-ase) activity. Both cis-PT-ases utilize farnesyl diphosphate (FPP) as a starter for polyprenyl diphosphate (dolichol backbone) formation. The products of the Rer2 and Srt1 proteins consist of 14-17 and 18-23 isoprene units, respectively. In this work we demonstrate that deletion or overexpression of SRT1 up-regulates the activity of Rer2p and dolichol content. However, upon overexpression of SRT1, preferential synthesis of longer-chain dolichols and a decrease in the amount of the shorter species are observed. Furthermore, overexpression of the ERG20 gene (encoding farnesyl diphosphate synthase, Erg20p) induces transcription of SRT1 mRNA and increases the levels of mRNA for RER2 and DPM1 (dolichyl phosphate mannose synthase, Dpm1p). Subsequently the enzymatic activity of Rer2p and dolichol content are also increased. However, the amount of Dpm1p or its enzymatic activity remain unchanged.
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