Recombinant CYP Bactosomes: versatile formats for many ADME applications
Recombinant CYPs are commonly used in ADME-Tox studies as in vitro tools, mostly for reaction phenotyping, enzyme inhibition screening, clearance and metabolite ID.
“Research has shown that rCYPs expressed in E. coli (Bactosomes) showed greater enzymatic activity compared with the equivalent enzymes expressed in insect cells (Supersomes). Similarly, most Bactosomes show greater turnover numbers when compared with the equivalent Supersomes. These results demonstrate that human rCYPs expressed in E. coli are frequently more active than the corresponding rCYPs expressed in insect cells» says Michael Voice from CYPEX.
If this statement from a CYP expert has encouraged you to learn more about CYPEX E. coli expressed CYPs (Bactosomes), this post should provide you with some clues on this interesting product range and how their flexibility in terms of formats can be an asset to suit your need.
What are the main features of Bactosomes?
- Patented E. coli expression system
- Excellent batch-to-batch consistency
- High freezze/Thaw stability
- Patented production method for native, or minimally modified P450’s
- Robust P450 activity levels
- Extensive batch-specific data (Km, Vmax, linearity with time and enzyme, at Km)
- Comprehensive selection, including modified-reductase choices and controls
- ISO9001:2009 compliant production and quality control
- 10-30 minute linearity over time
What are the benefits of Bactosomes?
- Bactosomes utilize native P450 and better represent human enzymes
- Available with and without b5
- Different levels of reductase allow for high and low activities
- Reductase Bactosomes are optimized for metabolism
- Low Reductase Bactosomes developed specifically for inhibition assays
- Bactosomes exhibit the best linearity over time compared to competitor rCYPs
- Least expensive commercial option
- Extensive panel of P450s and Phase II expressed enzymes available
- Only commercial source for recombinant dog P450s
- Custom rCYPs available
Broad Linearity range: high and low reductase Bactosomes +/- b5 cytochrome
High and low reductase Bactosomes: The activity of many cytochrome P450s can vary significantly with the amount of NADPH P450 reductase available. Reductase (R) and High Reductase (HR) Bactosomes exhibit a limited linearity of substrate turnover with time. This is due to the fact that they contain high levels of NADPH P450 reductase, in turn creating higher activity. They may be better suited for metabolite profiling and reaction phenotyping. Low Reductase Bactosomes contain lower levels of NADPH P450 reductase, enhancing the linearity of substrate turnover with time making Bactosomes a perfect system for inhibition studies and more comparable to activities found in vivo. Whether you choose low or high reductase Bacotosomes will therefore depend on your application.
… with or without b5 Cytochrome: Addition of cytochrome b5 to bactosomes preparations has various effect depending on the assay conditions and which isoform is being used. For example use of CYP2E1 with cytochrome b5 is highly recommended as it reduces the Km for the reaction, but not necessarily always with CYP3A4 as the effect of b5 seems to disappear when the assay conditions are set to give high activity (that may not apply for a different substrate).
Flexible formats: EasyCYP standard vial size & Classic Bactosomes
Cypex EasyCYPs are a more convenient form of the bactosomes as they are standardised for CYP concentration (1nmol/mL) and protein concentration (10mg/mL). This means that, for routine assays, the volume to be added does not need to be recalculated for each different batch.
An example of all available formats discussed above is provided below for human CYP3A4 and human CYP-reductase coexpressed in Escherichia coli, with high or low reductase levels, supplemented or not with purified human cytochrome b5, each as standard or EasyCYP formats:
- Reductase with cytochrome b5:CYP3A4BR and CYP3A4BR EasyCYP
- Low reductase with cytochrome b5:CYP3A4BLR and CYP3A4BLR EasyCYP
- Reductase:CYP3A4R and CYP3A4R EasyCYP
- Low reductase:CYP3A4LR and CYP3A4LR EasyCYP
If you can’t find the CYP you are looking for, don’t hesitate to contact me. Customization can be possible!