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. 2021 Jul 26;108(9):1735–1751. doi: 10.1016/j.ajhg.2021.07.001

Figure 3.

Figure 3

Multiplexed measurement of CYP2C9 activity via VAMP-seq

(A) Using VAMP-seq, we expressed a CYP2C9 library in HEK293T cells such that each variant was expressed as an eGFP fusion, resulting in a range of fluorescence according to variant stability. We then flow sorted cells into bins and sequenced them to determine relative variant abundance.

(B) Flow cytometry of CYP2C9 WT (red), destabilizing variant p.Arg335Trp (∗11, blue), and CYP2C9 eGFP fusion library expressed in HEK293T cells (black outline). Smoothed histograms of eGFP:mCherry ratios are shown. Approximate quartile bins for sorting shown are at the top.

(C) Stacked histogram of abundance score colored by type of variant. Abundance score of p.Arg335Trp (∗11) is shown as a point.

(D) Scatterplot and linear regression of individually measured cell eGFP:mCherry ratios for 12 CYP2C9 variants versus VAMP-seq-derived abundance scores for the same variants. Error bars show standard error for abundance scores and standard error for individually determined eGFP:mCherry ratio (n = 2 replicates).