Difference between revisions of "Part:BBa K5477038"

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CYP3A4-pGAL1/10-POR detox module against a wide array of contaminants

The CYP3A4-pGAL1/10-POR composite part consists the CYP3A4 enzyme BBa_K5477020 and cytochrome P450 oxidoreductase (POR) BBa_K5477022 to form a detoxification module. The pGAL1/10 bidirectional promoter drives the co-expression of CYP3A4 and POR in opposite directions. CYP3A4, a phase I enzyme, is responsible for oxidizing around 50% of all clinically used drugs (1) (2) (3) (4). POR provides the electrons required for these oxidation reactions by transferring electrons from NADPH to CYP3A4 (5).

This composite part was cloned using the method of USER-cloning into YCp-H. YCp-H is a centromeric plasmid used in yeast that includes a HIS3 marker, allowing for selection in histidine auxotrophic yeast strains. Like other CEN plasmids, YCp-H contains a CEN sequence, ensuring that the plasmid replicates and segregates similarly to yeast chromosomes. This results in a low copy number (typically one to two copies per cell), providing stable maintenance of the plasmid.

Results

Objective: To evaluate the detoxification system by incubating it with specific contaminants and performing LC-MS analysis to determine whether new compounds are produced.

Methodology: The detoxification module CYP1A1-pGAL1/10-POR with UDPD-pGAL1/10-UGT1A1 was induced with galactose and incubated with PCB. Following overnight incubation, each detoxification system was centrifuged in Eppendorf tubes to separate the cells from the supernatant. The supernatant, henceforth referred to as the “media” samples, was collected. Absolute ethanol was added to the cell pellets, which were vortexed with micro glass beads to lyse the cells. The mixtures were centrifuged to separate cellular debris from the lysate, and the resulting supernatant was collected as the “pellet” samples.

All samples were filtered prior to being loaded into LC-MS glass vials.

Result: We did not have optimized LC-MS methods available for detecting highly hydrophobic compounds, even with the use of a Reverse Phase Column. Additionally, it took some time to identify ethanol as a suitable solvent for the system available in the department. Therefore, the results presented here are based on data generated from a single run of the samples on the LC-MS system. A previous run, where DMSO was used as a solvent, was excluded from the experiment due to the presence of multiple nonspecific peaks.

Here, we tried to test CYP3A4 system on bith PCB and BPA. We decided to do so as CYP3A4 P450 enzyme is seen to work on a diverse substrates. However, here we were unable to find any unique peaks. Here the system was incubated with 10 µM of PCB#3 and BPA each.

Figure 1 Base Peak Chromatogram for C3A4 detox system. Pellet, media, control samples are included.

Sequence and Features

References

1. Bardal SK, Waechter JE, Martin DS. Chapter 6 - Pharmacogenetics. In: Bardal SK, Waechter JE, Martin DS, editors. Applied Pharmacology [Internet]. Philadelphia: W.B. Saunders; 2011. p. 53–8. Available from: https://www.sciencedirect.com/science/article/pii/B9781437703108000063

2. Guengerich, F.. (2008). Cytochrome P450 and Chemical Toxicology. Chemical research in toxicology. 21. 70-83. 10.1021/tx700079z.

3. Klyushova LS, Perepechaeva ML, Grishanova AY. The Role of CYP3A in Health and Disease. Biomedicines. 2022 Oct 24;10(11):2686. doi: 10.3390/biomedicines10112686. PMID: 36359206; PMCID: PMC9687714.

4. Lynch T, Price A. The effect of cytochrome P450 metabolism on drug response, interactions, and adverse effects. Am Fam Physician. 2007;76(3):391-396.

5. Pandey AV, Flück CE. NADPH P450 oxidoreductase: structure, function, and pathology of diseases. Pharmacol Ther. 2013;138(2):229-254. doi:10.1016/j.pharmthera.2013.01.010