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Engineering stability into Escherichia coli secreted Fabs leads to increased functional expression
Stephen J Demarest1, Gang Chen, Bruce E Kimmel
1Department of Protein Therapeutics, Diversa Corp. 4955 Directors Place San Diego, CA 92121, USA. stephen.demarest@biogenidec.com
Protein Engineering, Design & Selection : PEDS
|May 5, 2006
Summary
Improving antibody fragment (Fab) expression in E. coli involves enhancing intrinsic stability. This study identified key mutations to increase Fab stability, leading to better yields and functionality for therapeutic antibody development.
Area of Science:
- Biotechnology
- Protein Engineering
- Immunology
Background:
- Recombinant expression of antibody fragments like Fabs and scFvs in E. coli shows significant variability.
- Poor expression is often associated with intrinsic instability and misfolding of these antibody domains.
Purpose of the Study:
- To investigate the link between Fab stability and expression levels in E. coli.
- To develop a novel strategy for stabilizing a poorly expressing human anti-tetanus toxoid Fab.
- To identify specific mutations that enhance Fab stability and function.
Main Methods:
- Saturation mutagenesis was performed on 45 selected residues within the Fab.
- Automated screening identified variants with increased thermal denaturation midpoint temperature (TM).
- Mutations were combined to maximize Fab stability.
Main Results:
- Over 4000 library members were screened, identifying 11 key residue positions for stabilization, primarily in the Fv region.
- Combined mutations increased the Fab's TM to 92°C.
- Enhanced stability correlated with increased Fab yields and improved protein folding and functionality.
Conclusions:
- Increasing Fab intrinsic stability is a viable strategy to improve recombinant expression in E. coli.
- Optimized Fabs showed enhanced thermostability in IgG1 format in mammalian cells, though expression levels were unaffected.
- This approach has implications for producing stable, functional antibody domains and therapeutic antibodies.