Improved methodology for refolding functional TGF-beta superfamily ligands

The TGF-beta Superfamily is composed of secreted cell-regulatory proteins which have biologically diverse roles in embryonic development, body patterning, tissue differentiation and proliferation, maintenance of pluripotency, and influence of cell fate in embryonic and other stem cell systems. There are nearly forty TGF-beta Superfamily ligands in the mammalian genome, however only about a dozen have been successfully refolded into their dimeric active conformations after expression in E. coli. The goal of this study was to add to the body of currently refoldable functional ligands. This report presents a novel screening approach to identify optimal conditions for the refolding of several TGF-beta family members, since correct three-dimensional structure is critical to protein folding and function. We also incorporated the previously successful "1B" strategy to modify and improve dimer yield of five ligands. Initial small scale screening of ligand-condition combinations was used to identify a condition that allowed most efficient refolding for each ligand. For the best ligand-condition combinations, the refolding volume was increased for purification studies and to test ligand activity in cell based assays. Various refolding conditions proved to be highly effective in increasing recovery of properly folded and functional protein. By developing conditions that optimize refolding of TGF-beta family ligands we produced never-before properly refolded and active TGF-beta ligands. This screening methodology can be applied to other TGF-beta Superfamily homodimers and heterodimers in future studies.