title: Evaluation of GFP tag as a screening reporter in directed evolution of a hyperthermophilic beta-glucosidase.
abstract: By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP's fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable beta-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.
authors.affiliation: Department of Genetics, Universidade de São Paulo, Piracicaba, SP, Brazil. andreolima@hotmail.com
authors.forename: André O S
authors.initials: AO
authors.lastname: Lima
authors.forename: Diane F
authors.initials: DF
authors.lastname: Davis
authors.forename: Gavin
authors.initials: G
authors.lastname: Swiatek
authors.forename: James K
authors.initials: JK
authors.lastname: McCarthy
authors.forename: Dinesh
authors.initials: D
authors.lastname: Yernool
authors.forename: Aline A
authors.initials: AA
authors.lastname: Pizzirani-Kleiner
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Eveleigh
mesh_terms: D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D006090:Gram-Negative Bacteria / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D001617:beta-Glucosidase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001617:beta-Glucosidase