Bacterial expression of two human aryl sulfotransferases

The effect of replacing a single codon in the N-terminal of human aryl sulfotransferase (HAST) 1 and 3 with one that is more commonly found in E. coli genes was assessed. The pKK233-2 E. coli expression vector was employed and the polymerase chain reaction (PCR) was used to introduce the 5' nucleotide substitution, at the same time maintaining the fidelity of the amino acid sequence. The data indicates that this change had a minimal effect on protein production, subcellular localization or, in the case of HAST3, catalytic activity. In general, the pKK233-2 E. coli vector has been less than optimal for expressing human sulfotransferase cDNAs. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved

Strigolactones and shoot branching: what is the real hormone and how does it work?

OnlinePublThere have been substantial advances in our understanding of many aspects of strigolactone regulation of branching since the discovery of strigolactones as phytohormones (Gomez-Roldan et al., 2008; Umehara et al., 2008). These include further insights into the network of phytohormones and other signals that regulate branching, as well as deep insights into strigolactone biosynthesis, metabolism, transport, perception, and downstream signalling. In this review, we provide an update on recent advances in our understanding of how the strigolactone pathway co-ordinately and dynamically regulates bud outgrowth and pose some important outstanding questions that are yet to be resolved.Elizabeth A. Dun, Philip B. Brewer, Elizabeth M.J. Gillam, Christine A. Beveridg

Facile production of minor metabolites for drug development using a CYP3A shuffled library

Metabolic profiling of new drugs is limited by the difficulty in obtaining sufficient quantities of minor metabolites for definitive structural identification. Biocatalytic methods offer the potential to produce metabolites that are difficult to synthesize by traditional medicinal chemistry. We hypothesized that the regioselectivity of the drug metabolizing cytochrome P450s could be altered by directed evolution to produce minor metabolites of drugs in development. A biocatalyst library was constructed by DNA shuffling of four CYP3A forms. The library contained 11±4 (mean±SD) recombinations and 1±1 spontaneous mutations per mutant. On expression in Escherichia coli, 96% of mutants showed detectable activity to at least one probe substrate. Using testosterone as a model drug-like substrate, mutants were found that preferentially formed metabolites produced in only trace amounts by parental forms. A single 1.6. L batch culture of one such mutant enabled the facile isolation of 0.3. mg of the minor metabolite 1Β-hydroxytestosterone and its ab initio structural determination by 1D- and 2D-NMR spectroscopy