Optimizing random heteropolymers to improve protein folding in cell-free synthesis

Membrane proteins play key roles in biological activities, and represent one of most prevailing drug targets. Functional and structural analysis of membrane proteins remain intriguing but challenging due to the protein misfolding and aggregation. Here, we show that four-monomer random heteropolymers (RHPs) can assist membrane protein folding during translation in cell free synthesis. Two types of membrane proteins, Aquaporin Z (water channel,helices) and OmpT (protease, barrel) were examined, and demonstrated tunable polymer performance by controlling the composition of RHPs. The results indicated that the extent of folding assistance of various RHPs for the membrane proteins was different due to the chemical heterogeneity along the length of RHP chains. An in-house program for chemical heterogeneity analysis was developed to investigate the sequences of various RHPs and proteins. The current data suggests that RHPs act as artificial chaperones and that their ability to aid in the proper folding of membrane proteins can be easily tuned by changing their composition.