CARDILLO Alejandra Beatriz
congresos y reuniones científicas
Hyoscyamine 6b-hydroxylase expression in Saccharomyces cerevisiae
CARDILLO, ALEJANDRA BEATRIZ; MARTINEZ, CAROLINA; GIULIETTI, ANA MARIA
Congreso; SAIB 2005; 2005
Saccharomyces cerevisiae is a host of choice for the expression of heterologous proteins due to its ability to produce recombinant proteins correctly folded and modified at the posttranslational level. In addition, this organism has been classified as Generally Regarded As Safe (GRAS). The reasons mentioned above made yeast a very attractive organism to work with on biotechnological processes. Hyoscyamine 6b-hydroxylase (H6H) catalyzes the conversion of hyoscyamine into scopolamine. The latest is widely used as a pharmaceutical. We isolated the h6h mRNA from different Brugmansia candida organs. In this work we report the cloning and expression of H6H in S. cerevisiae for future industrial applications. The gene was cloned into the yeast pYES2.1-TOPO TA expression vector. The constructions were confirmed by sequencing. Recombinant yeast clones were isolated by cultures in YNBD medium containing histidine, leucine and tryptophan without uracil. The expression of the h6h cloned was induced by changing the dextrose carbon source for galactose. The expression of the enzyme as a fusion protein allowed us to detect it by Western blot using antibodies against V5 epitope. The protein is expressed after 4 hours induction. The functionality of the cloned enzyme will be assayed by scopolamine HPLC detection.