Table 2.
Yield of soluble protein and kinetic parameters and for the various hybrid dimers of 11β-HSD1 purified from the bacterial expression system
| Construct |
||||
| pRSF-1 (His tag) | pET-51 (Strep tag) | Yield (mg l−1) | kcat (min−1) | Km (μM) |
| WT | WT | 0.047 ± 0.003 | 2.29 ± 0.18 | 7.62 ± 1.52 |
| R137C | WT | 0.008 ± 0.001 | 1.73 ± 0.15 | 14.72 ± 2.81 |
| K187N | WT | 0.008 ± 0.004 | — | — |
The kcat and Km values for the WT-His/WT-Strep heterodimer construct are not significantly different from the previous homodimeric WT constructs, although the yield of the heterodimer is lower. Heterodimeric R137C/WT constructs have decreased kcat values and increased Km values. There also is a significant reduction in the yield of soluble protein compared with the WT/WT equivalent. Heterodimeric K187/WT constructs have no detectable enzyme activity, in addition to a significant reduction in the yield of soluble protein. Yield values are given in mg of protein per L of LB broth ± SEM (n = 3).