Table 3.
AV activity of R2E increases upon storage
| Preparation and form | Date refolded | Refolding conditions | Ligand binding | Initial AV activity, nM | Final AV activity, nM | Increased activity, fold |
|---|---|---|---|---|---|---|
| 1, GST-RE | 4/23/98 | 1 | N.P. | 126 | 26 | 5 |
| 2, R2E | 1/27/99 | 2 | + | Undetectable | 340 | — |
| 3, R2E | 2/24/99 | 1 | N.P. | Undetectable | 13 | — |
| 4, GST-R2E | 3/18/99 | 2 | + | 960 | 54 | 18 |
| 5, GST-R2E | 3/18/99 | 1 | N.P. | Undetectable | 90 | — |
| 6, GST-R2E | 5/14/99 | 2 | + | 85 | 3 | 28 |
| 7, GST-R2E | 5/14/99 | 2 | + | 1400 | 9 | 155 |
| 8, GST-R2E | 5/01/00 | 2 | − | Undetectable | Undetectable | — |
Samples 6 and 7 were stored frozen until February 27, 2000 and at 4°C until determination of final AV activity. All others were maintained aseptically at 4°C until assayed. Proteins were prepared as either GST-fusion proteins or unconjugated in the pET15b vector. Initial activity was determined within 7 days from refolding. Final AV activity and ligand-binding activity were determined on June 20, 2000. N.P., assay was not performed because preparations were in Tris buffer. Refolding conditions: 1, 50 mM Tris⋅HCl (pH 8.0), 100 mM NaCl; 2, PBS, 10% (vol/vol) glycerol, 1 mM reduced glutathione 5 mM oxidized glutathione.