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Engineering soluble proteins for structural genomics

Nature Biotechnology volume 20, pages 927–932 (2002)Cite this article

Abstract

Structural genomics has the ambitious goal of delivering three-dimensional structural information on a genome-wide scale. Yet only a small fraction of natural proteins are suitable for structure determination because of bottlenecks such as poor expression, aggregation, and misfolding of proteins, and difficulties in solubilization and crystallization. We propose to overcome these bottlenecks by producing soluble, highly expressed proteins that are derived from and closely related to their natural homologs. Here we demonstrate the utility of this approach by using a green fluorescent protein (GFP) folding reporter assay to evolve an enzymatically active, soluble variant of a hyperthermophilic protein that is normally insoluble when expressed in Escherichia coli, and determining its structure by X-ray crystallography. Analysis of the structure provides insight into the substrate specificity of the enzyme and the improved solubility of the variant.

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Figure 1: Protein solubility and fluorescence measurements.
Figure 2: The NDP kinase fold.
Figure 3: The nucleotide binding site.
Figure 4: The dimer interface.

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Acknowledgements

We thank Leon Flaks for his assistance in performing data collection. We also thank James Jett, Andrew Bradbury, and Kathleen Sandman for review of the manuscript, and the National Institutes of Health and University of California Campus Laboratory Collaboration Program for generous support.

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Authors and Affiliations

  1. Bioscience Division, MS-M888, Los Alamos National Laboratory, Los Alamos, 87545, NM

    Jean-Denis Pédelacq, Chang-Yub Kim, Beom-Seop Rho, Min S. Park, Thomas C. Terwilliger & Geoffrey S. Waldo

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

    Elaine C. Liong & Joel Berendzen

  3. University of Rochester, Rochester, 14627, NY

    Emily Piltch

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  1. Jean-Denis Pédelacq
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Correspondence to Geoffrey S. Waldo.

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The authors have filed pending US patent application 09/410,889, “Method for determining and improving peptide/protein solubility.”

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Pédelacq, JD., Piltch, E., Liong, E. et al. Engineering soluble proteins for structural genomics. Nat Biotechnol 20, 927–932 (2002). https://doi.org/10.1038/nbt732

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