Development and characterization of an automated high throughput screening method for optimization of protein refolding processes

Abstract

Optimization of protein refolding parameters by automated, miniaturized, and parallelized high throughput screening is a powerful approach to meet the demand for fast process development with low material consumption. In this study, we validated methods applicable on a standard liquid handling robot for screening of refolding process parameters by dilution of denatured lysozyme in refolding buffer systems. Different approaches for the estimation of protein solubility and folding were validated concerning resolution and compatibility with the robotic system and with the complex buffer and protein structure composition. We established an indirect method to assess soluble lysozyme concentration independent of matrix effects and protein structure varieties by automated separation of aggregated protein, resolubilization, and measurement of absorption at 280 nm. Using this nonspecific solubility assays the correlation between favorable parameters for high active and soluble lysozyme yields were evaluated. An overlap of good refolding buffer compositions was found provided that the redox environment was controlled with redox reagents. In addition, the need to control unfolding conditions like time, temperature, lysozyme, and dithiothreitol concentration was pointed out as different feedstocks resulted in different refolding yields.

Bioscreening and expression of a camel anti-CTGF VHH nanobody and its renaturation by a novel dialysis–dilution method

“…During the dialysis method, the protein was retained in the dialysis bag and the proteins would start to refold while the denatured chemicals diffused outward. However, the dialysis was a complicated and time-consuming method requiring numerous repeats to optimize the refolding conditions and buffer composition, and generally taken multiple steps of dialysis (Jungbauer and Kaar 2007 ; Zilinskas and Sereikaite 2011 ; Berg et al 2012 ). As we known, during the protein refolding, the major obstruction came from the self-aggregation (Zilinskas and Sereikaite 2011 ; Gautam et al 2012 ).…”

Parameter Optimization in High-Throughput Testing for Structural Materials

“…Industrialization is particularly characterized by the use of high-throughput systems. These enable a faster acquisition of data from experiments with the lowest possible material consumption, by means of automation, parallelization, and minimization of processes [10]. Accordingly, high-throughput methods are increasingly used in material development.…”

Downstream process development strategies for effective bioprocesses: Trends, progress, and combinatorial approaches

“…An IB refolding process for a fully automated robotic workstation for lysozyme and different proteins from E. coli were introduced by Berg et al. , Vincentelli et al. , and Dechavanne et al.…”

“…The entire process of IB unfolding, protein refolding, and isolation of the correctly folded species is not straightforward, making HTPD the method of choice for designing such processes. An IB refolding process for a fully automated robotic workstation for lysozyme and different proteins from E. coli were introduced by Berg et al [82], Vincentelli et al [83], and Dechavanne et al [84]. The protein unfolding and refolding process included investigation of different pH ranges, salts, as well as additives like DTT, glutathione, folding enhancers (chaperones), glycerol, PEG, and co-factors.…”