Figure 3.

(A) Western blots of lysates of yeast cells expressing two copies of the CYP2D6-wt, CYP2D6.1, CYP2D6.10, and CYP2D6-C proteins, from the HIS3 and URA3 chromosomal loci of the strain YY7. Total protein (10 μg) from ∼1 × 10⁶ cells, obtained after lysis of the four strains (expressing 2 copies of each the four CYP2D6 alleles), was probed with a CYP2D6 antibody (panel A; Santa Cruz Biotechnology, sc-130366) and a β-actin antibody (panel B; Proteintech, 60008-1-Ig). Lane 1, CYP2D6-wt; lane 2, CYP2D6.1; lane 3, CYP2D6.10; lane 4, CY2D6-C; and lane 5, 0.3 pmol of CYP2D6-wt Sacchrosomes (i.e., microsomal CYP2D6-wt enzyme, isolated from baker’s yeast; CYP Design Ltd), as a positive control. (B,C) Calibration of CYP2D6 activities produced by 1/ × 10⁷ cells that express CYP2D6-wt (2D6-wt), CYP2D6-C (2D6-C), and CYP2D6.10 (2D6.10) proteins (B), using a standard curve (C) drawn using specific amounts (1, 1.5, 2, and 3 pmol) of CYP2D6 Supersomes (Corning, #456217); CYP enzymes from Corning are widely considered to be the benchmark in the area of recombinant human CYP microsomal enzymes.