Increased Fab thermoresistance via VH-targeted directed evolution
| Autor: | Jennifer L. Johnson, Raquel L. Lieberman, Kevin C. Entzminger, Jennifer A. Maynard, Jeongmin Hyun |
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| Rok vydání: | 2015 |
| Předmět: |
Models
Molecular Phage display Protein Conformation Molecular Sequence Data Immunoglobulin Variable Region Mutagenesis (molecular biology technique) Bioengineering Complementarity determining region Biology medicine.disease_cause Biochemistry Single Domain Antibodies Libraries and Innovations Protein structure Antigen Peptide Library medicine Humans Amino Acid Sequence Antigens Peptide library Molecular Biology Mutation Base Sequence Protein Stability Temperature Directed evolution Molecular biology Mutagenesis Directed Molecular Evolution Immunoglobulin Heavy Chains Biotechnology |
| Zdroj: | Protein Engineering Design and Selection. 28:365-377 |
| ISSN: | 1741-0134 1741-0126 |
| DOI: | 10.1093/protein/gzv037 |
| Popis: | Antibody aggregation is frequently mediated by the complementarity determining regions within the variable domains and can significantly decrease purification yields, shorten shelf-life and increase the risk of anti-drug immune responses. Aggregation-resistant antibodies could offset these risks; accordingly, we have developed a directed evolution strategy to improve Fab stability. A Fab-phage display vector was constructed and the VH domain targeted for mutagenesis by error-prone PCR. To enrich for thermoresistant clones, the resulting phage library was transiently heated, followed by selection for binding to an anti-light chain constant domain antibody. Five unique variants were identified, each possessing one to three amino acid substitutions. Each engineered Fab possessed higher, Escherichia coli expression yield, a 2–3°C increase in apparent melting temperature and improved aggregation resistance upon heating at high concentration. Select mutations were combined and shown to confer additive improvements to these biophysical characteristics. Finally, the wild-type and most stable triple variant Fab variant were converted into a human IgG1 and expressed in mammalian cells. Both expression level and aggregation resistance were similarly improved in the engineered IgG1. Analysis of the wild-type Fab crystal structure provided a structural rationale for the selected residues changes. This approach can help guide future Fab stabilization efforts. |
| Databáze: | OpenAIRE |
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