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Protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein

Nature Biotechnology volume 19, pages 131–136 (2001)Cite this article

Abstract

Protein misfolding is the basis of a number of human diseases and presents an obstacle to the production of soluble recombinant proteins. We present a general method to assess the solubility and folding of proteins in vivo. The basis of this assay is structural complementation between the α- and ω- fragments of β-galactosidase (β-gal). Fusions of the α-fragment to the C terminus of target proteins with widely varying in vivo folding yield and/or solubility levels, including the Alzheimer's amyloid β (Aβ) peptide and a non-amyloidogenic mutant thereof, reveal an unambiguous correlation between β-gal activity and the solubility/folding of the target. Thus, structural complementation provides a means of monitoring protein solubility/misfolding in vivo, and should find utility in the screening for compounds that influence the pathological consequences of these processes.

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Figure 1: An in vivo solubility assay based on structural complementation.
Figure 2: Colony color correlates with fusion protein solubility.
Figure 3: Target protein solubility.
Figure 4: Correlation of β-gal activity with fusion protein solubility and folding.
Figure 5: In vivo aggregation/insolubility of Aβ/α-fragment fusion proteins.

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Acknowledgements

We thank S. Muallem for helpful discussions and encouragement, M. Corboy for critical review of the manuscript, and members of our laboratories for helpful comments. This work was supported by a research grant from the Haberecht Foundation to W.C.W., a startup grant from Columbia University to J.F.H., and grants from the National Institutes of Health–National Institute of Diabetes and Digestive and Kidney Diseases and the Cystic Fibrosis Foundation to P.J.T.

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Authors and Affiliations

  1. Department of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, 75235, TX

    W. Christian Wigley, Rhesa D. Stidham, Nathan M. Smith & Philip J. Thomas

  2. Graduate Program in Molecular Biophysics, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, 75235, TX

    Rhesa D. Stidham & Philip J. Thomas

  3. Department of Biological Sciences, Columbia University, New York, 10027, NY

    John F. Hunt

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Correspondence to Philip J. Thomas.

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Wigley, W., Stidham, R., Smith, N. et al. Protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein. Nat Biotechnol 19, 131–136 (2001). https://doi.org/10.1038/84389

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