Table 2.
F1 fentanyl hapten (BLI) and free fentanyl (Competitive ELISA and DSF) binding data for mammalian expressed and purified WT and mutant JGFN4 recombinant homodimers
| JGFN4 dimer mutant ID | BLI KD (M) | Comp. ELISA IC50 (M) | DSF KDa (M) | |||
|---|---|---|---|---|---|---|
| WT | 1.07E-08 | ±8.10E-10 | 2.26E-05 | ±5.11E-06 | 1.10E-03 | ±8.91E-05 |
| A74Y | 1.20E-08 | ±6.51E-11 | 2.48E-05 | ±2.93E-06 | 4.35E-04 | ±3.01E-05 |
| A74W | 1.63E-08 | ±1.13E-09 | 3.61E-05 | ±1.14E-05 | 3.23E-04 | ±9.33E-06 |
| R53H | No Binding | 1.78E-05 | ±2.49E-06 | 8.67E-04 | ±1.54E-04 | |
| N76D | 8.46E-09 | ±6.54E-10 | 4.43E-06 | ±1.78E-07 | 8.65E-05 | ±1.03E-06 |
| N76H | 1.36E-08 | ±1.46E-10 | 7.95E-06 | ±4.30E-07 | 8.01E-05 | ±3.27E-06 |
| A74Y/N76D | 5.80E-09 | ±2.05E-09 | 4.14E-06 | ±3.90E-07 | 4.26E-05 | ±1.54E-06 |
| S29Y/N76D | 5.45E-09 | ±4.38E-10 | 5.35E-06 | ±5.46E-07 | 6.46E-06 | ±2.00E-07 |
| S29Y/A74Y/N76D | 5.43E-09 | ±5.01E-10 | 9.58E-06 | ±2.50E-06 | 5.60E-07 | ±4.00E-08 |
| M34A | No Binding | No Binding | 8.74E-04 | ±1.39E-05 | ||
| K71A | No Binding | No Binding | 7.36E-04 | ±9.60E-03 | ||
| N32A | No Binding | No Binding | 3.80E-02 | ±3.73E-02 | ||
Highest affinity binder(s) for each measure denoted by italics. BLI error reported as 1 standard deviation. Competitive ELISA and DSF error reported as ± SEM of four and three replicates per sample, respectively. See Supporting Information for full data sets.
a
As determined at 600 uM free fentanyl concentration.