title: A practical teaching course in directed protein evolution using the green fluorescent protein as a model.
abstract: 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.
authors: Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade São Paulo, São Paulo, Brazil.
authors: Roberto
authors: R
authors: Ruller
authors: Rafael
authors: R
authors: Silva-Rocha
authors: Artur
authors: A
authors: Silva
authors: Maria Paula
authors: MP
authors: Cruz Schneider
authors: Richard John
authors: RJ
authors: Ward
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000193:education / Q000379:methods; D019020:Directed Molecular Evolution / Q000295:instrumentation / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D037402:Hydrozoa / Q000378:metabolism; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D018794:Problem-Based Learning / Q000379:methods; D011487:Protein Conformation; D015202:Protein Engineering / Q000295:instrumentation / Q000379:methods*; D013663:Teaching / Q000379:methods*
chemical_list: D050505:Mutant Proteins; D049452:Green Fluorescent Proteins