Cytochrome P450 reductase expression systems

Although mammalian CYPs are attractive candidates for use as commercial biocatalysts, many functional characteristics limit the opportunities to exploit such a system. Association of the enzymes with membranes prevents easy extraction and purification and limits the opportunities to produce useful recombinant enzymes by cloning the relevant genes for expression in microbial systems. All P450s have a porphyrin-haem active site that requires a second protein to reduce the iron component, often cytochrome P450 reductase or... 

Evaluation of approach to predict the contribution of multiple cytochrome P450s in drug metabolism using relative activity factor effects of the differences in expression levels of NADPH-cytochrome P450 reductase and cytochrome b(5) in the expression system and the differences in the marker activities. Journal of Pharmaceutical Sciences, 91, 952—963. 

The expression levels (as P450 content) discussed above assume adequate, but not necessarily saturating, cytochrome P450 reductase levels, and reasonable linearity of the system (i.e. linear metabolism in microsomes or lysates for at least 30 min). If a system is linear for shorter time periods, higher expression levels are needed if the system is linear for longer periods of time, lower expression levels are adequate. 

Fig. 1. The testosterone 6j8-hydroxylase activity (expressed as turnover number) for cDNA-expressed CYP3A4 using yeast, bacteria and mammalian cell systems. Yeast-based expression with coexpressed human cytochrome P450 reductase and human cytochrome or native, yeast cytochrome P450 reductase and cytochrome bs (Peyronneau et al., 1992). E. co/i-GSH E. co/i-based expression where the enzyme was purified and reconstituted with and without the addition of glutathione (Gillam et al., 1993). Human lymphoblast-based expression with endogeneous cytochrome P450 reductase (Crespi et al., 1991a) and with coexpression of cytochrome P450 reductase cDNA (C. Crespi, unpublished observation). Vaccinia virus (vv)/HepG2 cell expression data from Buters et al. (1994).

Andersen JF, Utermohlen JG, Feyereisen R (1994) Expression of housefly CYP6A1 and NADPH-cytochrome P450 reductase in Escherichia coli and reconstitution of an insecticide-metabolizing P450 system. Biochemistry 33 2171-2177... 

In a recent investigation to develop novel cytochrome P450 biocatalysts, DNA shuffling was used to produce chimeric cytochrome P450s mutants with enhanced biocatalytic activities, which were then co-expressed with NADPH-cytochrome reductase in E. coli to form an efficient system, in this case demonstrated to be effective for indole oxidation [69]. 

Preparations containing a single P450 isozyme are available as either expression systems or purified, reconstituted enzymes. The P450s have been expressed in bacterial, yeast, insect, and mammalian cells (8). Most of these enzymes can be used in the membranes in which they are expressed. However, in order to obtain adequate enzyme activity for most expression systems, it is necessary to supplement the membranes with reductase and in some cases cytochrome b5. This is accomplished by either supplementing the membranes with purified coenzymes or by coexpression of the coenzymes. Alternatively, the P450 enzymes can be purified and reconstituted with coenzymes into artificial membranes. 

Approaches to the prediction of hepatic metabolism using a model system such as the Dundee P450 System are based on assumptions about the veracity of the transposed cytochrome P450 oxido-reductase complex when expressed in E. coli. So long as the investigator accepts the limits imposed by those assumptions, large numbers and types of compounds can be screened rapidly. 

In early work in this field, this point would have been the demonstration of the reaction of interest with an enzyme purified from tissue. Today P450 proteins are generally produced in recombinant systems and seldom purified from tissue sources. In routine practice in the pharmaceutical industry, new reactions are examined with a battery of the major recombinant human (liver) P450s, many of which are available from commercial sources. Systems used for expression include bacteria, yeast, baculovirus (-infected insect cells), and mammalian cells. The P450s need not be purified for these comparisons hut must have suitable provision for NADPH-P450 reductase in a crude system (and cytochrome... 

Nodate M, Kubota M, Misawa N (2006) Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp NCIMB 9784. Appl Microbiol Biotechnol 71 455 62... 

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