Evaluation of GFP tag as a screening reporter in directed evolution of a hyperthermophilic beta-glucosidase.
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.
Lima
André O S
AO
Department of Genetics, Universidade de São Paulo, Piracicaba, SP, Brazil. andreolima@hotmail.com
Davis
Diane F
DF
Swiatek
Gavin
G
McCarthy
James K
JK
Yernool
Dinesh
D
Pizzirani-Kleiner
Aline A
AA
Eveleigh
Douglas E
DE
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*
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001617:beta-Glucosidase