title: Directed evolution of stabilized IgG1-Fc scaffolds by application of strong heat shock to libraries displayed on yeast.
abstract: We have constructed IgG1-Fc scaffolds with increased thermal stability by directed evolution and yeast surface display. As a basis a new selection strategy that allowed the application of yeast surface display for screening of stabilizing mutations in proteins of already high intrinsic thermal stability and T(m)-values up to 85°C was developed. Besides library construction by error prone PCR, strong heat stress at 79°C for 10min and screening for well-folded proteins by FACS, sorting rounds had to include an efficient plasmid DNA isolation step for amplification and further transfection. We describe the successful application of this experimental setup for selection of 17 single, double and triple IgG1-Fc variants of increased thermal stability after four selection rounds. The recombinantly produced homodimeric proteins showed a wild-type-like elution profile in size exclusion chromatography as well as content of secondary structures. Moreover, the kinetics of binding of FcRn, CD16a and Protein A to the engineered Fc-molecules was very similar to the wild-type protein. These data clearly demonstrate the importance and efficacy of the presented strategy for selection of stabilizing mutations in proteins of high intrinsic stability within reasonable time.
authors.affiliation: University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Traxlmayr
authors.forename: Maximilian
authors.initials: M
authors.lastname: Faissner
authors.forename: Gerhard
authors.initials: G
authors.lastname: Stadlmayr
authors.forename: Christoph
authors.initials: C
authors.lastname: Hasenhindl
authors.forename: Bernhard
authors.initials: B
authors.lastname: Antes
authors.forename: Florian
authors.initials: F
authors.lastname: Rüker
authors.forename: Christian
authors.initials: C
authors.lastname: Obinger
mesh_terms: D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D019020:Directed Molecular Evolution; D018869:Heat-Shock Response; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D020125:Mutation, Missense; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017452:Receptors, IgG / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology; D013816:Thermodynamics
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D019151:Peptide Library; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins