| Autor: |
Wang Y; University of Groningen, Department of Drug Design, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands., van Oosterwijk N; University of Groningen, Department of Drug Design, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands., Ali AM; University of Groningen, Department of Drug Design, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands., Adawy A; University of Groningen, Department of Drug Design, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands., Anindya AL; University of Groningen, Department of Drug Design, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands., Dömling ASS; University of Groningen, Department of Drug Design, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands., Groves MR; University of Groningen, Department of Drug Design, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands. M.R.Groves@rug.nl. |
| Abstrakt: |
Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which "helper" molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the "helper" molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency. |
