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Directed evolution of the surface chemistry of the reporter enzyme β-glucuronidase

Nature Biotechnology volume 17, pages 696–701 (1999)Cite this article

Abstract

The use of the Escherichia coli enzyme β-glucuronidase (GUS) as a reporter in gene expression studies is limited due to loss of activity during tissue fixation by glutaraldehyde or formaldehyde. We have directed the evolution of a GUS variant that is significantly more resistant to both glutaraldehyde and formaldehyde than the wild-type enzyme. A variant with eight amino acid changes was isolated after three rounds of mutation, DNA shuffling, and screening. Surprisingly, although glutaraldehyde is known to modify and cross-link free amines, only one lysine residue was mutated. Instead, amino acid changes generally occurred near conserved lysines, implying that the surface chemistry of the enzyme was selected to either accept or avoid glutaraldehyde modifications that would normally have inhibited function. We have shown that the GUS variant can be used to trace cell lineages in Xenopus embryos under standard fixation conditions, allowing double staining when used in conjunction with other reporters.

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Figure 1: Screen for glutaraldehyde-resistant β-glucuronidase (GUS) function (sequence from top left).
Figure 2: Detection of glutaraldehyde-resistant GUS activity.
Figure 3: GUS catalytic activity as a function of aldehyde concentration.
Figure 4: Homology mapping of amino acid substitutions that confer aldehyde resistance.
Figure 5: Expression of GUSAR in Xenopus embryos.
Figure 6: Multiple marker staining of Xenopus embryos.

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Acknowledgements

We thank the Office of Naval Research for funding. I.M. was supported by a National Science Foundation/Alfred P. Sloan Postdoctoral Research Fellowship in Molecular Evolution (DBI-9750002). We thank Dr. Mary Berlyn of the E. coli Genetic Stock Center for sending us strain GMS407 and Ms. Sabine Bell for synthesizing oligonucleotides. Finally, we thank members of the Ellington group and Dr. William Wu of Ambion for helpful discussion.

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Author notes

  1. John B. Wallingford

    Present address: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, 94720

  2. Neeraj K. Surana

    Present address: Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO, 63108

Authors and Affiliations

  1. Institute of Cellular and Molecular Biology, ICMB A4800/MBB 3.424, 26th and Speedway, University of Texas, Austin, 78712, TX

    Ichiro Matsumura, Neeraj K. Surana & Andrew D. Ellington

  2. Department of Zoology, Center for Developmental Biology, University of Texas, Austin, 78712, TX

    John B. Wallingford & Peter D. Vize

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  1. Ichiro Matsumura
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Correspondence to Andrew D. Ellington.

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Matsumura, I., Wallingford, J., Surana, N. et al. Directed evolution of the surface chemistry of the reporter enzyme β-glucuronidase. Nat Biotechnol 17, 696–701 (1999). https://doi.org/10.1038/10910

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