A Novel Design of a Translation Coupling-RNA Scaffold System to Improve the Efficiency of Molecular Chaperone on Recombinant Proteins Solubilization

Inclusion bodies formation upon the overexpression of recombinant proteins in Escherichia coli is among the major obstacles in therapeutic proteins production and metabolic engineering.  Numerous approaches have been attempted for minimizing the formation of misfolded and aggregated recombinant proteins, such as the use of solubility-enhancing tags, the modification of physicochemical conditions, and the overexpression of molecular chaperones.  Among those approaches, the overexpression of molecular chaperones is considered as the most economical and time-efficient approach for producing soluble recombinant proteins in E. coli.  However, the efficacy of this strategy is still limited by a low exposure of molecular chaperones to the newly synthesized recombinant proteins due to the crowded and viscous environment of E. coli cytoplasm.  To further improve the chaperone-mediated solubilization of recombinant proteins in E. coli, we designed a novel RNA scaffold system in which the 3’-UTR of the recombinant protein-encoding mRNA serves as a scaffold for localizing the molecular chaperone in proximity with the newly synthesized proteins.  In our system,  a molecular chaperone DnaJ was fused with an RNA binding domain that specifically binds a unique RNA sequence in an engineered RNA hairpin loop structure on the 3’-UTR of the recombinant protein-encoding mRNA.  This design can prevent the formation of inclusion bodies by promoting the rapid interaction between molecular chaperones and newly synthesized recombinant proteins.  As expected, our RNA scaffold system successfully increased the solubility of selected aggregation-prone proteins overproduced in E. coli (UDP-6-glucose-dehydrogenase, anti p21-Ras ScFv, and anti p21-Ras ScFv fused with a cell penetrating peptide), which was better than the solubility increment obtained by their simple co-expression with DnaJ molecular chaperone.  Our RNA scaffold system would provide a valuable tool for the production of recombinant proteins in soluble and active forms in E. coli with minimal post-production process.