Rapid and convenient biotransformation procedure for human drug metabolizing enzymes using permeabilized fission yeast cells.
The development of convenient assays for the in vitro study of drug metabolizing enzymes (DMEs) such as cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) greatly facilitates metabolism studies of candidate drug compounds and other xenobiotics. We have developed and optimized an experimental approach that combines the advantages of recombinant expression in yeast with a microsomal-like biotransformation and thus allows for rapid and convenient enzymatic assays. Recombinant strains of the fission yeast Schizosaccharomyces pombe have previously been demonstrated to functionally express human CYPs and UGTs. Permeabilization of such cells with Triton X-100 results in the formation of enzyme bags, which can be used as biocatalysts. This protocol describes the preparation of such enzyme bags (3 h) and their application in enzyme activity assays (4 h) utilizing either pro-luminescent substrates and luminescence measurements or non-luminescent substrates and liquid chromatography coupled to mass spectrometry (LC-MS). Both applications provide practical tools for investigating CYP and UGT reactions in vitro without the need for additional sophisticated instrumentation or expertise.
Sharma S, Durairaj P, Bureik M