A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables

Abstract

Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which “helper” molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the “helper” molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency.

Theory and applications of differential scanning fluorimetry in early-stage drug discovery

“…One year later, colleagues in our group expanded the DGR approach by investigating the refolding agent arginine and other additives in systematic buffer screens (Wang et al 2017). Arginine has been widely used to suppress protein aggregation in refolding, and it can slow or prevent protein association reactions via weak interactions with the targets (Baynes et al 2005;Arakawa et al 2007), distinct from Fig.…”

“…This has been shown to increase the success rates of protein crystallization in past decades (Huynh and Partch 2015). More recently, DSF has also been applied to the challenge of sample preparation, with two publications demonstrating that suitable screening approaches can be used to identify and optimize sample refolding buffers-allowing significantly cheaper access to the amounts of protein sample required to support high-throughput screening campaigns (Biter et al 2016;Wang et al 2017). Finally, a very recent development has shown that DSF is able to provide reliable data in complex solutions, such as unpurified chemical reactions.…”

Refolding Proteins from Inclusion Bodies using Differential Scanning Fluorimetry Guided (DGR) Protein Refolding and MeltTraceur Web

“…Protein folding (or refolding) is affected by numerous variables such as temperature, ionic environment, small molecule additives, pH, etc. (Biter et al, 2016;Burgess, 2009;Clark, 1998;Middleberg, 2002;Wang et al, 2017). Unfortunately, conditions that favor proper protein folding over alternative non-productive pathways, notably protein aggregation, cannot yet be predicted a priori, dictating that refolding conditions must be determined empirically (Goldberg & Rainer Rudolph, 1991).…”

“…This is tedious and time‐consuming, due to the fact that protein folding is affected by numerous factors including salts and ions in solution, pH, additives and buffer composition, temperature, etc. (Anselment et al., ; Biter, de la Pena, Thapar, Lin, & Phillips, ; Wang et al., ). Thus, protein refolding has been referred to as a “method of last resort.” (Graslund, Nordlund, Weigelt, & Gunsalus, )…”

“…With a workflow similar to that used in crystallography, DGR uses both commercially available and custom buffer screens to rapidly test numerous refolding conditions in a high-throughput fashion (Biter et al, 2016). Suitable refolding conditions discovered via DGR can be ramped up to preparative scale to produce proteins for various purposes (Clark, 1998(Clark, , 2001Middleberg, 2002;Wang et al, 2017).…”