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

Abstract

Differential Scanning Fluorimetry Guided Refolding (DGR) is a simple methodology that can be used to rapidly screen for and identify conditions capable of accurately refolding protein preparations, such as those obtained from Escherichia coli inclusion bodies. It allows for the production in E. coli of functional proteins that would otherwise require far more expensive production methods. This unit describes how to set up a DGR refolding assay, perform DGR refolding trials in microplate format, use MeltTraceur Web software to interactively analyze the resulting data, scale‐up protein production via refolding, and lastly, validate that the protein is properly folded. © 2018 by John Wiley & Sons, Inc.

Three Essential Resources to Improve Differential Scanning Fluorimetry (DSF) Experiments

“…Data analysisInaccessibility of robust, efficient data analysis remains a significant and widespread bottleneck for DSF users. Recent reports have presented both scripts and websites for the analysis of DSF data[52][53][54][55][56][57] , we found that two substantial bottlenecks remained unaddressed. First, there remains a need for tools to efficiently, and flexibly visualize the effects of experimental variables to facilitate data interpretation and optimizations.…”

Differential scanning fluorimetry as a measure of functionality of refolded anti-malarial antibody fragments

“…It has been used to identify buffer conditions in which the purified proteins are stable for biophysical studies including crystallization, and to qualitatively determine binding to small molecules, peptides and nucleic acids (25, 26, 27, 28). DSF has been reported to screen and identify refolding conditions in which the protein is folded optimally, reflected by a low initial fluorescence and sigmoidal unfolding transition of the melt profiles (29, 30). 4B7-Db showed a melting temperature of 59.07 ± 1°C determined using nanoDSF, with a low initial fluorescence and sigmoidal melt profile.…”

Application of temperature-responsive HIS-tag fluorophores to differential scanning fluorimetry screening of small molecule libraries

“…Beyond screening, there is a significant diversity of manners in which the DSF assay is applied, including buffer and crystallization formulation and binding mechanism studies. Extending these applications, DSF was recently applied to improving refolding conditions of protein after denaturing purifications ( Biter et al, 2016 ; Wang et al, 2017 ; Lee et al, 2019 ; Ronzetti et al, 2022 ). The thermal shift technique has also been applied to more complex protein-protein interactions and for the quantification of overexpressed protein in lysates ( Seo et al, 2014 ; Shao et al, 2020 ).…”