Heterologous Expression of Grapefruit Clones PGT3 and PGT9 in Yeast and Screening of Recombinant Protein for Activity
The wide diversity of plant secondary products results from different modifications undergone during biosynthesis, including glucosylation. These modification reactions result in production of the compounds actually found in plants and to unique chemical and biochemical properties, including some bitter compounds in grapefruit. While the presence of a PSPG box motif allows for identification of a clone as a putative glucosyltransferase (PGT), diversity of GT primary structures makes it difficult to accurately assign specific function. Our approach is to identify and isolate putative GT clones, express them heterologously, and biochemically characterize the proteins. Eleven putative GT clones have been isolated from Citrus paradise and some have been biochemically characterized. The current hypothesis being tested is that PGT3 and PGT9 clones are plant secondary product GTs. Due to issues with inclusion bodies when using E. coli, proteins were expressed in Pichia pastoris using the pPICZA vector. Recombinant protein expression was confirmed by Western blot and proteins were enriched by IMAC. Over 30 flavonoid and simple phenolic substrates, representing many compounds found in grapefruit, were screened for activity with PGT3 and PGT9 proteins. No significant activity was found and the biochemical function of the proteins encoded by these clones will be further investigated.
London, ON, Canada
Wamucho, Anye; Hayford, Deborah; and McIntosh, Cecelia A.. 2012. Heterologous Expression of Grapefruit Clones PGT3 and PGT9 in Yeast and Screening of Recombinant Protein for Activity. Poster presentation. 51st Annual Meeting of the Phytochemical Society of North America, London, ON, Canada. http://www.psna-online.org/PSNA2012-Program&Abstracts.pdf