title: An enhanced approach for engineering thermally stable proteins using yeast display.
abstract: Many biotechnology applications require the evolution of enhanced protein stability. Using polymerase chain reaction-based recovery of engineered clones during the screen enrichment phase, we describe a yeast display method capable of yielding engineered proteins having thermal stability that substantially exceeds the viability threshold of the yeast host. To this end, yeast-enhanced green fluorescent protein destabilized by dual-loop insertion was engineered to possess a substantially enhanced resistance to thermal denaturation at 70°C. Stabilized proteins were secreted, purified and found to have three- to six-fold increased resistance to thermal denaturation. The validated method enables yeast display-based screens in previously inaccessible regions of the fitness landscape.
authors: Department of Chemical and Biological Engineering,  University of Wisconsin-Madison, Madison, WI 53706,  USA.
authors: Tej V
authors: TV
authors: Pavoor
authors: Jean A
authors: JA
authors: Wheasler
authors: Viraj
authors: V
authors: Kamat
authors: Eric V
authors: EV
authors: Shusta
mesh_terms: D000595:Amino Acid Sequence; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013696:Temperature
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins