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Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic β-Glucosidase

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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 β-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.

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Acknowledgments

AOSL was supported through The Brazilian National Council for Scientific Research and Technological Development (CNPq) and The Brazilian Foundation and Coordination for Graduate Student Improvement (CAPES). This study was supported by a USDA National Research Initiative Competitive Grants Program grant 97-35503-4557, NJ Marine Science Consortium Grant B/T-12, a McIntire-Stennis grant 0181520 and the NJ Agricultural Experiment Station.

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  1. André O. S. Lima

    Present address: Centro de Ciências Tecnológicas da Terra e do Mar, Universidade do Vale do Itajaí, R. Uruguai, 458, Itajaí, SC, 88302-202, Brazil

Authors and Affiliations

  1. Department of Genetics, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Av. Pádua Dias 11, Piracicaba, SP, 13418-900, Brazil

    André O. S. Lima & Aline A. Pizzirani-Kleiner

  2. Department of Biochemistry and Microbiology, School of Environmental and Biological Science, Rutgers University, 76 Lipman Drive, New Brunswick, NJ, 08901-8525, USA

    Diane F. Davis, Gavin Swiatek, James K. McCarthy, Dinesh Yernool & Douglas E. Eveleigh

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Lima, A.O.S., Davis, D.F., Swiatek, G. et al. Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic β-Glucosidase. Mol Biotechnol 42, 205–215 (2009). https://doi.org/10.1007/s12033-009-9152-1

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