title: Generating thermal stable variants of protein domains through phage display.
abstract: Often in protein design research, one desires to generate thermally stable variants of a protein or domain. One route to identifying mutations that yield domains that remain folded and active at a higher temperature is through the use of directed evolution. A library of protein domain variants can be generated by mutagenic PCR, expressed on the surface of bacteriophage M13, and subjected to heat, such that the unfolded forms of the domain, showing reduced or no binding activity, are lost during subsequent affinity selection, whereas variants that still retain binding to their target are selected and enriched with each subsequent round of affinity selection. This approach takes advantage of the fact that bacteriophage M13 particles are heat stable and resistant to many proteases and protein denaturants. We present the application of this general approach to generating thermally stable variants of a eukaryotic peptide-binding domain. The benefits of producing such variants are that they typically express at high levels in Escherichia coli (30-60 mg/L shake flask) and remain soluble in solution at higher concentrations for longer periods of time than the wild-type form of the domain. The process of library generation and screening generally requires about one month of effort, and yields variants with >10 °C increase in thermal stability, as measured in a simple fluorescence-based thermal shift assay. It is anticipated that thermally stable variants will serve as excellent scaffolds for generating affinity reagents to a variety of targets of interest.
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, 845 W. Taylor St., 3240 SES-MC 066, Chicago, IL 60607-7060, USA.
authors.forename: Kritika
authors.initials: K
authors.lastname: Pershad
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kay
mesh_terms: D017104:Bacteriophage M13 / Q000235:genetics; D061505:Cell Surface Display Techniques; D017076:Computer-Aided Design; D004797:Enzyme-Linked Immunosorbent Assay; D011489:Protein Denaturation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D013696:Temperature