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Rapid protein-folding assay using green fluorescent protein

Nature Biotechnology volume 17, pages 691–695 (1999)Cite this article

Abstract

Formation of the chromophore of green fluorescent protein (GFP) depends on the correct folding of the protein. We constructed a "folding reporter" vector, in which a test protein is expressed as an N-terminal fusion with GFP. Using a test panel of 20 proteins, we demonstrated that the fluorescence of Escherichia coli cells expressing such GFP fusions is related to the productive folding of the upstream protein domains expressed alone. We used this fluorescent indicator of protein folding to evolve proteins that are normally prone to aggregation during expression in E. coli into closely related proteins that fold robustly and are fully soluble and functional. This approach to improving protein folding does not require functional assays for the protein of interest and provides a simple route to improving protein folding and expression by directed evolution.

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Figure 1: Solubility of proteins expressed in E. coli is highly correlated with fluorescence of E. coli expressing corresponding GFP fusions.
Figure 2: Improved solubility of nonfusion (black bars) and GFP fusion fluorescence (crosshatched bars) for (A) gene V (C33T) and (B) bullfrog H-subunit ferritin during rounds of directed evolution.
Figure 3: GFP fusion fluorescence, nonfusion solubility, and activity of ferritin variants.
Figure 4: Fraction of indicated ferritin variant that is overexpressed in soluble form in E. coli at 37°C (crosshatched bars) or 27°C (black bars).

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Acknowledgements

We wish to thank Sorel Fitz-Gibbon and Jeffrey Miller (UCLA) for P. aerophilum DNA, sequence data, and BLAST searches. We also thank Thomas Peat, James Jett, Scott Peterson, Chia-Hwa Chang, and Raymond Nanni for helpful discussions and review of the manuscript, and the NIH and LDRD program LANL for generous support.

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Authors and Affiliations

  1. Structural Biology Group, MS-M888, Los Alamos , NM 87545

    Geoffrey S. Waldo & Thomas C. Terwilliger

  2. Biophysics Group, MS-P244, Los Alamos National Laboratory , Los Alamos, NM 87545

    Joel Berendzen

  3. University of New Mexico, Albuquerque, 87131, NM

    Blake M. Standish

Authors
  1. Geoffrey S. Waldo
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  2. Blake M. Standish
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  4. Thomas C. Terwilliger
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Correspondence to Geoffrey S. Waldo.

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Waldo, G., Standish, B., Berendzen, J. et al. Rapid protein-folding assay using green fluorescent protein. Nat Biotechnol 17, 691–695 (1999). https://doi.org/10.1038/10904

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