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A practical teaching course in directed protein evolution using the green fluorescent protein as a model
Author(s) -
Ruller Roberto,
SilvaRocha Rafael,
Silva Artur,
Cruz Schneider Maria Paula,
Ward Richard John
Publication year - 2011
Publication title -
biochemistry and molecular biology education
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.34
H-Index - 39
eISSN - 1539-3429
pISSN - 1470-8175
DOI - 10.1002/bmb.20430
Subject(s) - green fluorescent protein , aequorea victoria , fluorescence , protein engineering , fluorophore , mutagenesis , mutant , fluorescent protein , directed evolution , site directed mutagenesis , biochemistry , biophysics , biology , computational biology , chemistry , gene , enzyme , physics , quantum mechanics
Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from Aequorea victoria by a random mutagenesis strategy using error‐prone polymerase chain reaction. Screening of bacterial colonies transformed with random mutant libraries identified GFP variants with increased fluorescence yields. Mapping the three‐dimensional structure of these mutants demonstrated how alterations in structural features such as the environment around the fluorophore and properties of the protein surface can influence functional properties such as the intensity of fluorescence and protein solubility. Biochemistry and Molecular Biology Education Vol. 39, No. 1, pp. 21‐27, 2011