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.
Submitter: Shu-Ching Ou
Submission Date: Nov. 15, 2018, 12:04 p.m.
|Number of data points||168|
|Proteins||Immunoglobulin heavy constant gamma 1|
|Assays/Quantities/Protocols||Experimental Assay: Thermal Stability: Tm_3 ; Experimental Assay: Thermal Stability: Tm_2 ; Experimental Assay: Thermal Stability: Tm_1 ; Derived Quantity: Thermal Stability: ΔTm_3 ; Derived Quantity: Thermal Stability: ΔTm_2 ; Derived Quantity: Thermal Stability: ΔTm_1 ; Derived Quantity: SD of Thermal Stability: Tm_3 ; Derived Quantity: SD of Thermal Stability: Tm_2 ; Derived Quantity: SD of Thermal Stability: Tm_1|
|Libraries||Mutants of IgG1-Fc|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|
|100.0||Immunoglobulin heavy constant gamma 1||P0DOX5||IGG1_HUMAN|
|100.0||Immunoglobulin heavy constant gamma 1||P01857||IGHG1_HUMAN|
|93.7||Immunoglobulin heavy constant gamma 1||P01859||IGHG2_HUMAN|
|93.7||Immunoglobulin heavy constant gamma 1||P01861||IGHG4_HUMAN|
|94.2||Immunoglobulin heavy constant gamma 1||P01860||IGHG3_HUMAN|