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Computer-based engineering of thermostabilized antibody fragments |
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| Authors: | Jiwon Lee Bryan S. Der Christos S. Karamitros Wenzong Li Nicholas M. Marshall Oana I. Lungu Aleksandr E. Miklos Jianqing Xu Tae Hyun Kang Chang-Han Lee Bing Tan Randall A. Hughes Sang Taek Jung Gregory C. Ippolito Jeffrey J. Gray Yan Zhang Brian Kuhlman George Georgiou Andrew D. Ellington |
| Affiliation: | 1. Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire;2. Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina;3. Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas;4. Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas;5. U.S. Army Combat Capabilities Development Command Chemical Biological Center, APGEA, Aberdeen, Maryland;6. Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Massachusetts;7. Biopharmaceutical Chemistry Major, School of Applied Chemistry, Kookmin University, Seongbuk-gu, Seoul, Republic of Korea;8. U.S. Army Research Laboratory, Austin, Texas Applied Research Laboratories, The University of Texas at Austin, Austin, Texas;9. Department of Biomedical Science, Graduate School of Medicine, Korea University, Seoul, Republic of Korea |
| Abstract: | We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (Tm) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher Tm compared to the parental scFv and importantly to retain full antigen binding activity after 2 hr of incubation at 70°C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction. |
| Keywords: | antibody engineering thermostable antibodies Rosetta scAb scFv |