A Novel Chimeric Avidin with Increased Thermal Stability Using DNA Shuffling

• DOI: 10.1371/journal.pone.0092058
• Publication Date: 2014-03-14T20:45:47Z
• Authors: Barbara Taskinen; Tomi T. Airenne; Janne Jänis; Rolle Rahikainen; Mark S. Johnson; Markku S. Kulomaa; Vesa P. Hytönen
• Keywords: Avidin; Biopanning; DNA shuffling; Protein Engineering; Streptavidin

Avidins are a family of proteins widely employed in biotechnology. We have previously shown that functional chimeric mutant can be created from avidin and avidin-related protein 2 using methodology combining random mutagenesis by recombination selection tailored biopanning protocol (phage display). Here, we report the crystal structure one selected characterized forms, A/A2-1. The was solved at 1.8 Å resolution revealed fold not affected shuffled sequences. also supports observed physicochemical properties mutant. Furthermore, improved screening to select for avidins with slower dissociation rate biotin than were earlier. This resulted A/A2-B, which showed increased thermal stability as compared A/A2-1 parental proteins. especially evident conditions extreme pH differential scanning calorimetry. In addition, amino acid sequence structural comparison mutants led rational design A/A2-B I109K. mutation further decreased yielded an increase stability.