| Optimizing Protein Stability and Function - Impact of ThermoFluor on Protein Overexpression and Primary Screening |
|
Presentation: 2C04 Session: Advancing Targets to HTSJames Kranz and Matthew Todd, Johnson & Johnson Pharmaceutical Research and Development, L.L.C. Presenting Author: James Kranz, Johnson & Johnson PRD - USA Understanding effects of solution conditions on both protein stability and function can provide critical insights into HTS screening results and optimizing conditions for protein purification. Thorough characterization of assay conditions facilitates interpretation of compound mechanisms arising from HTS and may lead to relevant secondary counterscreens. ThermoFluor® is a high throughput protein thermal stability assay that directly measures stability changes due to equilibrium binding of compounds. Protein stability varies with solution conditions, either due to a binding event (e.g. Mg2+ binding) or due to a bulk solvent effect (change in water activity). Solution effects may also affect measured affinity, particularly when binding is pH dependent. Standardized experimental setup and a 384-well format enable rapid, array-based characterization of varied solution conditions, allowing a rational approach to evaluate conditions for protein purification, crystallization, primary screening, in examining functional differences among variations in protein constructs, and in maximizing longevity and reproducibility in protein preparations. |