Journal
JOURNAL OF SEPARATION SCIENCE
Volume 35, Issue 22, Pages 3149-3159Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/jssc.201200306
Keywords
High throughput screening; High throughput process development; Protein analytics; Protein refolding
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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.
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