Towards high affinity carbohydrate-binding proteins: Directed evolution of murine galectin-3

Journal Article (Journal Article)

Towards a better understanding of the molecular basis of affinity, a directed evolution of murine galectin-3 (G3) was initiated to produce mutants with improved affinity for lactose and N-acetyllactosamine relative to the wild-type protein. A series of N-terminal truncations were developed to facilitate incorporation of the 35 kDa protein into a phage-display construct. Analysis of the various assemblies revealed that all such deletions produced protein unsuitable for use in directed evolution studies. Following fusion of the full-length galectin to p3 of filamentous phage, three libraries were constructed and biopanned for increased affinity for lactose. The first two libraries, of 1 × 10 and 1 × 10 members, respectively, were assembled through a combination of error-prone PCR and DNA shuffling. A third library was constructed using a modified staggered extension protocol (StEP), but contained only 10 members. Mutants were also engineered site-specifically to test the role of key residues in or near the binding pocket. Analysis of the mutants by ITC identified one mutation (R158G) that produces a twofold increase in affinity for lactose and another that results in a sixfold increase in affinity for N-acetyllactosamine. Solid-phase binding analysis of phage for nonexpressing proteins indicated that two other mutants demonstrated increased binding to β-methyllactose relative to the wild-type protein. Together these studies validate the evolutionary approach and set the stage for the development of novel carbohydrate-binding proteins. 5 6

Full Text

Duke Authors

Cited Authors

  • Lundquist, JJ; Kiburz, BM; Wu, JK; Gibbs, KD; Toone, EJ

Published Date

  • December 3, 2002

Published In

Volume / Issue

  • 80 / 8

Start / End Page

  • 999 - 1009

International Standard Serial Number (ISSN)

  • 0008-4042

Digital Object Identifier (DOI)

  • 10.1139/v02-086

Citation Source

  • Scopus