Potentiation of the activity of Escherichia coli chaperone DnaJ by tailing hyper-acidic minipeptides.

The chaperone network plays an essential role in cellular protein homeostasis. However, some core components often coaggregate with misfolded proteins for sequestration and dysfunction, leading to abnormal cell proteostasis, aggregation-associated disorders, and poor solubility of overexpressed recombinant proteins. Among them, DnaJ or its ortholog, an obligate co-chaperone in the tripartite DnaK-DnaJ-GrpE system, is of more implications, probably due to its intrinsic propensity for aggregation. Herein, we potentiated the activity of Escherichia coli DnaJ by using hyper-acidified protein fusion strategy. We found DnaJ did possess only a moderate solubility that could be remarkably improved by fusing hyper-acidic minipeptides. Most importantly, we revealed the hyper-acidified DnaJ with a fusion tail could outperform its native form (significantly up to 2.1-fold) to enhance the solubility of target proteins and meanwhile appropriately impart them an elevated activity. These results suggest the hyper-acidified DnaJs can chaperone target proteins with correct folding into a truly soluble and active form. Moreover, we showed these hyper-acidified DnaJ variants could surpass its prototype to confer E. coli or yeast an enhanced heat tolerance, and DnaJ itself could be solubilized by its hyper-acidified fusion cognates. Finally, we discussed the overall mechanism for DnaJ activity potentiation mediated by hyper-acidic tailing fusion.