Directed evolution of the surface chemistry of the reporter enzyme beta-glucuronidase.

Published

Journal Article

The use of the Escherichia coli enzyme beta-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.

Full Text

Duke Authors

Cited Authors

  • Matsumura, I; Wallingford, JB; Surana, NK; Vize, PD; Ellington, AD

Published Date

  • July 1999

Published In

Volume / Issue

  • 17 / 7

Start / End Page

  • 696 - 701

PubMed ID

  • 10404164

Pubmed Central ID

  • 10404164

International Standard Serial Number (ISSN)

  • 1087-0156

Digital Object Identifier (DOI)

  • 10.1038/10910

Language

  • eng

Conference Location

  • United States