Overview
This lecture introduces phage display, a powerful technique using bacteriophages to identify proteins, especially antibodies, that bind to specific targets, revolutionizing drug discovery and protein engineering.
Introduction to Phage Display
- Phage display is a technique to display proteins or peptides on the surface of bacteriophages (viruses that infect bacteria).
- Invented in 1985 by George Smith, with significant contributions from Gregory Winter (Nobel Prize 2018).
- Allows screening and selection of proteins (such as antibodies) from vast combinatorial libraries.
How Phage Display Works
- Foreign DNA encoding a protein of interest is inserted into a phage coat protein gene, resulting in a fusion protein displayed on the phage surface.
- Millions of phage variants are created, each displaying a different protein segment.
- The phage library is exposed to an immobilized target; phages that bind are retained, non-binders are washed away.
- Bound phages are eluted (freed), amplified in bacteria, and subjected to iterative cycles for improved selection.
- DNA from selected phages is sequenced to identify binding proteins, which can be expressed and tested further.
Key Components and Techniques
- Commonly used phages for display are filamentous, such as M13, Fd, and F1, which have single-stranded circular DNA.
- Display is typically via the p3 or p8 coat protein; p3 (with about 5 copies/phage) is often preferred.
- Monovalent (single copy) and polyvalent (multiple copies) displays affect binding affinity and avidity measurements.
- Helper phages and phagemids are used for controlled expression and display of fusion proteins.
Applications in Antibody and Drug Discovery
- Used to identify and optimize antibodies, circumventing the need for animal immunization.
- Libraries can be made from human antibody genes for fully human therapeutic antibodies, as seen with drugs like Humira.
- Technique enables engineering of antibody fragments (e.g., scFv, Fab) for research and therapies.
- Reverse panning allows identification of antigens/epitopes recognized by specific antibodies.
Technical Considerations and Variations
- Multiple rounds of selection introduce mutations, enabling “molecular evolution” of stronger binders.
- Use of mutator bacterial strains can further increase diversity and speed up evolution.
- Fusion proteins often include only antibody variable regions for display efficiency.
Key Terms & Definitions
- Phage (Bacteriophage) — A virus that infects bacteria.
- Fusion Protein — A protein created by joining two genes, resulting in a single polypeptide.
- Combinatorial Library — A large collection of different proteins or peptides generated from random DNA sequences.
- Bio-panning — The process of selecting binding phages from a library by exposing them to an immobilized target.
- Antigen — A substance recognized and bound by an antibody.
- Epitope — The specific region of an antigen bound by an antibody.
- Fab, scFv — Engineered antibody fragments used for display and binding studies.
Action Items / Next Steps
- Review mechanisms of phage display selection and amplification.
- Read further on antibody engineering and therapeutic applications.
- Prepare for questions regarding different display strategies (monovalent vs. polyvalent).