An integrated approach to extreme thermostabilization and affinity maturation of an antibody
| Autor: | Ashley D. Berkebile, Audrey D. McConnell, Peter Bowers, Joseph C. Sheffer, John L. Macomber, David J. King, Steven S. Neben, Andy P. Chen, Robert A. Horlick, Irina P. Krapf, Vladimir Spasojevich |
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| Rok vydání: | 2012 |
| Předmět: |
Models
Molecular Hot Temperature Recombinant Fusion Proteins Antibody Affinity Mutagenesis (molecular biology technique) Somatic hypermutation Bioengineering Plasma protein binding Protein Engineering Biochemistry Affinity maturation Mice Antigen Antibody Specificity Animals Humans Transition Temperature Computer Simulation Protein Interaction Domains and Motifs Amino Acid Sequence Molecular Biology Levivirus Protein Unfolding biology Protein Stability Chemistry Protein engineering Complementarity Determining Regions In vitro Kinetics HEK293 Cells Amino Acid Substitution Immunoglobulin G Mutagenesis Site-Directed biology.protein Cystine Capsid Proteins Directed Molecular Evolution Antibody Cell Surface Display Techniques Monte Carlo Method Protein Binding Single-Chain Antibodies Biotechnology |
| Zdroj: | Protein Engineering Design and Selection. 26:151-164 |
| ISSN: | 1741-0134 1741-0126 |
| Popis: | Antibodies are important tools for a broad range of applications due to their high specificity and ability to recognize virtually any target molecule. However, in order to be practically useful, antibodies must be highly stable and bind their target antigens with high affinity. We present a combinatorial approach to generate high-affinity, highly stable antibodies through the design of stable frameworks, specificity grafting and maturation via somatic hypermutation in vitro. By collectively employing these methods, we have engineered a highly stable, high-affinity, full-length antibody with a T(m) over 90°C that retains significant activity after heating to 90°C for 1 h, and has ~95-fold improved antigen-binding affinity. The stabilized IgG framework is compatible with affinity maturation, and should provide a broadly useful scaffold for grafting a variety of complementarity-determining region loops for the development of stable antibodies with desired specificities. |
| Databáze: | OpenAIRE |
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