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

The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a report er in gene expression studies is limited due to loss of activity during tis sue fixation by glutaraldehyde or formaldehyde. We have directed the evolut ion of a GUS variant that is significantly more resistant to both glutarald ehyde and formaldehyde than the wild-type enzyme. A variant with eight amin o 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, amin o acid changes generally occurred near conserved lysines, implying that the surface chemistry of the enzyme was selected to either accept or avoid glu taraldehyde modifications that would normally have inhibited function. We h ave shown that the GUS variant can be used to trace cell lineages in Xenopu s embryos under standard fixation conditions, allowing double staining when used in conjunction with other reporters.