An improved strategy of TGFβ3 expression in Escherichia coli: Exploiting folding modulators for a switch from misfolded to folded form

Transforming growth factor beta 3 (TGFβ3) exhibits a complex native structure featuring the presence of multiple disulfide bonds forming the active dimer. Consequently, its heterologous expression in microbial system invariably leads to inclusion body (IB) formation. In this study, we observed an interesting phenomenon of switching a significant fraction of misfolded TGFβ3 to folded form by modulating the cellular protein folding machinery. We carried out co-expression experiments with chaperones and demonstrated the requirement of a coordinated action of DnaK-DnaJ-GrpE and GroESL, to achieve the native soluble conformation of TGFβ3, during over-expression in E. coli. The novelty of this study lies in the fact that orchestration of a group of chaperones to work in concert for efficient folding and assembly of TGFβ3-like cytokines has not been widely explored. Additionally, we have also demonstrated that presence of osmolytes (sorbitol or trehalose) in the growth media have an appreciable impact on the solubility of TGFβ3. We have further shown a synergism between the effects of molecular chaperone and osmolytes on the solubility of TGFβ3. We have confirmed the functionality of soluble TGFβ3 by performing binding interactions with its cognate receptor TβRII. Our study delineates the fact that an effective combination of chaperones or optimum concentration of compatible osmolyte, can efficiently abrogate competing aggregation pathways and help attain the native conformation of a cysteine rich cytokine in a facile manner.