Engineering robust chaperone substrates for refolding studies using the highly bioluminescent protein NanoLuc

Abstract

Chaperones play critical functions during protein de novo folding, refolding after denaturation and during translocation and degradation in all organisms. The Hsp70 chaperone system (DnaK/J/E in E. coli) has been studied over more than 40 years, yet its mechanism remains poorly understood. This is partly, due to the complexity and fragility of its most studied protein substrate, namely Firefly Luciferase (550 amino acids), which even in its native state is rather unstable and displays a complex co-translational-like folding behavior that obscures chaperone contributions to its refolding, following thermal or chemical denaturation.