(A) study on the effect of spatial organization of molecular chaperones on the solubilization of recombinant proteins in Escherichia coli

Abstract

Producing functional recombinant proteins in soluble form is a difficult-to-comprehend frontier in engineering bacterial hosts. Although a majority of recombinant proteins are amenable as substrates for the natural molecular chaperone-mediated solubilisation, due to limited availability of molecular chaperones in cells, not all recombinant proteins have the chance to be solubilized. In this study, the effect of spatial constraint between molecular chaperones of DnaK chaperone system in Escherichia coli with the translation system producing a protein of interest was evaluated. In order to achieve the spatial constraint of molecular chaperones and their substrates, two systems, namely chaperone recruiting mRNA scaffold (CRAS) and chaperone-substrate co-localized expression (CLEX) system were developed. In CRAS system, E. coli chaperone DnaJ is anchored to 3’ untranslated region of an mRNA encoding a target protein via fusion to KH, an RNA binding domain. While in CLEX system, the translation of target proteins are coupled with that of DnaJ using overlapping stop-start codon 5’-TAATG-3’. Significant solubility enhancement of selected aggregation-prone recombinant proteins, up to 95%, was observed in the application of both systems. The success of using the systems emphasizes the importance of coupling the translation and refolding processes in a spatial constrained manner.