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Protein Science (2004), 13:370-380. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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An automated in vitro protein folding screen applied to a human dynactin subunit

Christoph Scheich1,2, Frank H. Niesen1,3, Robert Seckler4 and Konrad Büssow1,2

1 Proteinstrukturfabrik, Heubnerweg 6, 14059 Berlin, Germany
2 Max Planck Institut fuer Molekulare Genetik, Ihnestrasse 73, 14195 Berlin, Germany
3 Institut fuer Medizinische Physik und Biophysik, Charité Universitaetsmedizin Berlin, 10117 Berlin, Germany
4 Institut für Biochemie und Biologie, Universitaet Potsdam, 14476 Golm, Germany

(RECEIVED September 17, 2003; FINAL REVISION October 21, 2003; ACCEPTED October 21, 2003)



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.

Keywords: Folding screen; refolding; inclusion bodies; hydrophobic interaction chromatography; tryptophan fluorescence spectroscopy; human dynactin; structural genomics

Abbreviations: 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 {beta}-D-1-thiogalactopyranoside • MDH, malate dehydrogenase (from pig heart mitochondria) • TEV, tobacco etch virus

Reprint requests to: Christoph Scheich, Proteinstrukturfabrik, Heubnerweg 6, Berlin 14059, Germany; e-mail: scheich{at}molgen.mpg.de; fax: 49-30-32639-2833.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03304604.


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