Development of a Cytochrome P450 Expression System with Biomass Scale-up, Streamlined Downstream Processing and Stability Modifications
Recombinant human P450s are important in preclinical studies to discover possible interactions between drugs when taking medicines. Drugs may be either inhibitors or substrates for P450s. Some P450s also play a major role in cancer and other diseases. Recombinant P450s offer better activity, availability and consistency than primary hepatocytes. In this thesis, a recombinant P450 expression system in yeast was developed with the aim of providing a cost-effective way of producing human P450 enzymes upon the yeast’s endoplasmic reticulum (ER). These ER-bound P450s are usually referred to as microsomal P450s. A yeast expression system was used to co-express several human P450s and their reductases, essential for P450 activities. Episomal and chromosomally integrating plasmids with auxotrophic markers were used to build the system. Gene expression was by either GAL1 promoter (galactose inducible) or ADH2 promoter (ethanol inducible). High level P450 expressions and activities were optimised by the effect of different growth media. It was found that by using different media conditions for recombinant protein expression, P450 activities could be improved. Efforts were made to produce P450 proteins in fermentors. For these larger-scale cultures, a cheaper, cost effective alternative to lyticase-mediated cell wall lysis was developed; it involves mechanical cell disruption- without degrading the microsomes. For large volumes, fractionation of microsomes by ultracentrifugation was unfeasible so a procedure was established that allows precipitation using polyethylene glycol (PEG). Recombinant P450s are extremely temperature-sensitive. They are stable only at minus 80°C and rapidly lose activity at room temperature. Lyophilisation is known to extend the shelf-life of recombinant proteins. Although lyophilisation is a harsh procedure which could shear the P450 proteins, from the microsomal membranes, it was found it could stabilise P450 activity so that the enzymes could be kept at room temperatures for weeks. The process was extensively optimised using a variety of different buffers and lyophilisation conditions and it was confirmed that: (a) Recombinant microsomal P450s produced from yeast, insect and bacterial cells could be stabilised using lyophilisation; (b) The lyophilised P450s could be plated out in 96-well microtitre plates and the plates could be kept at ambient temperature (+21°C) for weeks without losing activity, indicating that the process was robust; (c) The lyophilised enzymes and un-lyophilised samples behaved in the same way regarding their, potency towards known inhibitors, in fluorometric/ mass spectrometry assays and shipment to overseas destinations for P450 activity testing.
- PhD