Assessing Heterologous Expression of Hyoscyamine 6β-Hydroxylase – A Feasibility Study

The protein sequence of the hyoscyamine 6β-hydroxylase gene from Hyoscyamusniger was analysed in silico for its potential of heterologous expression. Therefore different parameters determining the proteins properties and structure in prokaryotic or eukaryotic protein expression systems were taken into account. In silico prediction of co- and post-translational modifications revealed 25 putative glycosylation sites, one of which reported to be a co-factor stabilizing residue in 2-oxoglutarate dependent dioxygenases. Potential protein solubility and degradation (PEST) motifs were also evaluated. Together with the calculated physico-chemical properties the results indicated reasonable solubility but potential instability of the protein in Escherichia coli and Saccharomyces cerevisiae. Further a synthetic h6h-gene was introduced into the prokaryotic or eukaryotic hostsEscherichia coli and Saccharomyces cerevisiae to determine protein expression. The protein could be expressed in both organisms, though stability was confirmed to be an issue.