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Keywords:

  • Folding screen;
  • refolding;
  • inclusion bodies;
  • hydrophobic interaction chromatography;
  • tryptophan fluorescence spectroscopy;
  • human dynactin;
  • structural genomics
  • CAB, bovine carbonic anhydrase II (from bovine erythrocytes);
  • CD, circular dichroism;
  • CTAB, hexadecyltrimethylammonium bromide;
  • DLS, dynamic light scattering;
  • DTT, dithiothreitol;
  • FTIR, Fourier-transform infrared;
  • GdmHCl, guanidine hydrochloride;
  • GSH, reduced glutathione, GSSG, oxidized glutathione;
  • HIC, hydrophobic interaction chromatography;
  • IPTG, isopropyl β-D-1-thiogalactopyranoside;
  • MDH, malate dehydrogenase (from pig heart mitochondria);
  • TEV, tobacco etch virus

Abstract

The preparation of proteins for structural and functional analysis using the Escherichia coli expression system is often hampered by the formation of insoluble intracellular protein aggregates (inclusion bodies). Transferring those proteins into their native states by in vitro protein folding requires screening for the best buffer conditions and suitable additives. However, it is difficult to assess the success of such a screen if no biological assay is available. We established a fully automated folding screen and a system to detect folded protein that is based on analytical hydrophobic interaction chromatography and tryptophan fluorescence spectroscopy. The system was evaluated with two model enzymes (carbonic anhydrase II and malate dehydrogenase), and was successfully applied to the folding of the p22 subunit of human dynactin, which is expressed in inclusion bodies in E. coli. The described screen allows for high-throughput folding analysis of inclusion body proteins for structural and functional analyses.