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

Authors

  • Annette Berg,

  • Stefan A. Oelmeier,

  • Jörg Kittelmann,

  • Florian Dismer,

  • Jürgen Hubbuch

    Corresponding author
    1. Karlsruhe Institute of Technology (KIT), Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe, Germany
    • Correspondence: Professor Jürgen Hubbuch, Karlsruhe Institute of Technology (KIT), Institute of Process Engineering in Life Sciences, Section IV:Biomolecular Separation Engineering, Engler-Bunte-Ring 1, Karlsruhe, Germany

      E-mail: jurgen.hubbuch@kit.edu

      Fax: +049-721-608-46240

    Search for more papers by this author

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.

Ancillary