Figure 1. Overview of DGR.

(a) The target protein, commonly obtained from E.coli inclusion bodies, is isolated. (b) The protein is then solubilized with a chaotropic agent such as guanidine. Sypro Orange dye is added, to facilitate the DSF assay, either at this step or following protein dilution. (c) Refolding is attempted by diluting the solubilized protein 20-fold into a multi-well plate containing buffers and additives, such as the PACT screen, to be tested for refolding using a DSF experiment. (d) The raw fluorescence melts from a 96 well DSF experiment are analyzed as described in reference (13). (e) The first derivative of (d) is calculated to determine T_M values. (f) T_M’s are subsequently mapped to the plate to assist in identifying conditions producing apparent melting transitions. Conditions for which no unfolding transition was apparent, and thus, T_M values could not be calculated, are shown in white. Conditions of the 96 well PACT screen, which is the used for all subsequent refolding trials in this manuscript, are shown in (f). The upper left quadrant of the screen varies pH, while the upper right quadrant tests the effects of various cations at diverse pH’s, and the bottom half of the screen tests the effects of anions at 3 different pH’s and while unbuffered. Figures 1a–c were drawn by KJP.