Construction of a Stability Landscape of the CH3 Domain of Human IgG1 by Combining Directed Evolution with High Throughput Sequencing
By M. Traxlmayr, Christoph Hasenhindl, M. Hackl, G. Stadlmayr, J. Rybka, N. Borth, J. Grillari, F. Rüker, C. Obinger. 2012
56 citations recorded by the source corpus.
One of the most important but still poorly understood issues in protein chemistry is the relationship between sequence and stability of proteins. Here, we present a method for analyzing the influence of each individual residue on the foldability and stability of an entire protein. A randomly mutated library of the crystallizable fragment of human immunoglobulin G class 1 (IgG1-Fc) was expressed on the surface of yeast, followed by heat incubation at 79 °C and selection of stable variants that still bound to structurally specific ligands. High throughput sequencing allowed comparison of the mutation rate between the starting and selected library pools, enabling the generation of a stability landscape for the entire CH3 domain of human IgG1 at single residue resolution. Its quality was analyzed with respect to (i) the structure of IgG1-Fc, (ii) evolutionarily conserved positions and (iii) in silico calculations of the energy of unfolding of all variants in comparison with the wild-type protein. In addition, this new experimental approach allowed the assignment of functional epitopes of structurally specific ligands used for selection [Fc γ‐receptor I (CD64) and anti-human CH2 domain antibody] to distinct binding regions in the CH2 domain.
Subfields: Molecular Biology, Radiology, Nuclear Medicine and Imaging
How researchers see this paper
The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.
In Molecular Biology
- Stanley Fields
- Douglas M. Fowler
- Jason J. Stephany
- Luciano A. Abriata
- Julia M. Shifman
- Timothy G. Palzkill
- M. dal Peraro
- Ben Lehner
In Radiology, Nuclear Medicine and Imaging
Citations in the corpus
- Protein Engineering by Combined Computational and In Vitro Evolution Approaches. (2016, background): “to identify the residues responsible for the stability of the CH3 domain of human IgG1 [54].”
- Measuring the activity of protein variants on a large scale using deep mutational scanning (2014, methodology): “For example, protein display has been used to conduct deep mutational scans of protein-peptide 4,8–11 , protein-protein 12–14 and protein-small molecule 15 interactions.”
- How Structural and Physicochemical Determinants Shape Sequence Constraints in a Functional Enzyme (2015, methodology): “These technologies have been applied to full scans of a few proteins in studies about tolerance to substitution and distribution of fitness effects [18–22] and to monitor directed evolution experiments [23,24].”