Experimental optimization of protein refolding with a genetic algorithm

Abstract

Refolding of proteins from solubilized inclusion bodies still represents a major challenge for many recombinantly expressed proteins and often constitutes a major bottleneck. As in vitro refolding is a complex reaction with a variety of critical parameters, suitable refolding conditions are typically derived empirically in extensive screening experiments. Here, we introduce a new strategy that combines screening and optimization of refolding yields with a genetic algorithm (GA). The experimental setup was designed to achieve a robust and universal method that should allow optimizing the folding of a variety of proteins with the same routine procedure guided by the GA. In the screen, we incorporated a large number of common refolding additives and conditions. Using this design, the refolding of four structurally and functionally different model proteins was optimized experimentally, achieving 74-100% refolding yield for all of them. Interestingly, our results show that this new strategy provides optimum conditions not only for refolding but also for the activity of the native enzyme. It is designed to be generally applicable and seems to be eligible for all enzymes.

Overview of refolding methods on misfolded recombinant proteins from Escherichia coli inclusion bodies

“…Other approachment of selecting refolding method was reported using in silico assistance using GA. This in silico approachment helps to guide refolding methods and screening of buffer condition, which supports comprehensive experimental design [13]. Otherwise, using RSM experimental design to evaluate refolding factors (pH, buffer, additives, and temperature) also helps in refolding screening.…”

“…Furthermore, using differential scanning fluorimetry (DSF)-guided protein refolding helps to evaluate refolding for large number of samples and rapid measurements. Moreover, in silico assistance using genetic algorithm (GA) guides in buffer and refolding condition determination for screening purposes [13]. As a result of refolding step, inactive protein was converted into its active form.…”

Advances in monitoring and control of refolding kinetics combining PAT and modeling

“…For an efficient refolding process with the dilution by fed-batch mode, several parameters need to be considered such as denaturant concentration (De Bernardez Clark 1998 ; Dong et al 2004 ; Tsumoto et al 2003 ), protein concentration, mixing intensity, reaction temperature, and buffer components (Anselment et al 2010 ; Eiberle and Jungbauer 2010 ) as well as dissolved oxygen and redox potential for proteins with disulfide bonds (Pizarro et al 2009 ). Furthermore, the process needs to be adequately monitored to achieve good product quality.…”

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

“…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, )…”