*4.2. Protein Engineering of CYP2C8\*1*/*\*2*/*\*3*/*R139K*/*K399R*

The CYP2C8 plasmid from Dr. Johnson was used directly for engineering the CYP2C8 variants. Plasmids were amplified and purified using a Qiagen Plasmid mini-prep kit (Valencia, CA, USA). The R139K and K399R single nucleotide substitutions were made using forward and reverse primers containing each mutation and an inserted BspQI restriction enzyme site (New England Biolabs) (Table S1). BspQI is a class II restriction enzyme that will create a unique sticky-end cut one nucleotide removed from the restriction enzyme site. The resulting gene of the R139K amplification and the K399R primers were then used to construct the CYP2C8\*3 gene. A single \*2 mutation was made similarly containing a single substitution at I269F. The PCR reaction consisted of 1 μM of forward and reverse primers in HF reaction buffer (New England Biolabs) containing 50 pg/μL CYP2C8-containg plasmid, 200 μM dNTPs, 5% DMSO, and Phusion DNA polymerase (10 U/mL). The PCR thermocycler was set to 95 ◦C for 3 min, 20 cycles (95 ◦C for 30 sec, 65 ◦C for 30 sec), and then 72 ◦C for 4 min. The mutated plasmids were then digested with BspQI and the resulting sticky ends were ligated to make complete plasmids. Chemically competent DH5α cells were transformed by heat shock at 42 ◦C for 45 min, and then set on ice. The addition of 1 mL of warm Super Optimal Broth (SOC) media was followed by shaking (250 rpm, 37 ◦C) for 1 hr. Cells were plated on an LB Amp plate to screen for the desired mutant plasmid. Mutant dsDNA was confirmed by DNA sequencing at the UIUC Core Sequencing Facility. Cells were co-transformed with pTGro7 plasmid containing the GroES-GroEL chaperonin system.

#### *4.3. Protein Expression and Purification of CYP2C8\*1*/*\*2*/*\*3*/*R139K*/*K399R*

All CYP2C8 proteins were expressed according to the protocol used in CYP2J2 expression, as previously described [41,44]. The protein concentrations were determined using a UV–vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) (<sup>ε</sup> <sup>=</sup> 108 mM−1·cm<sup>−</sup>1).
