Site directed mutagenesis as an efficient way to enhance structural and spectral properties of green fluorescence protein

Site directed mutagenesis is an efficient way of introducing desired mutations in proteins including Green Fluorescence Protein (GFP). The fluorescence of GFP is due to excitation of double bonds within the amino acid chain at positions 65-67 (Serine-Tyrosine-Glycine) and changes in these amino acid residues in or near these positions can produce mutant GFP with variant fluorescence and spectral properties. In this work, site directed mutagenesis was used to create desired mutations; F64L(replacement of phenylalanine 64 with leucine) and S65T (replacement of serine 65 with threonine) in wild type GFP converting it to enhanced GFP that was expressed by auto-induction. Mass spectroscopy and fluorimetry were used to determine the molecular mass and fluorescence intensity respectively of the mutant protein. Themutations have enhanced the structural and spectral properties of the protein.