▪ Amber codon TAG: supE strains (glutamic acid
codon), non-suppressor strains (stop codon)
▪ Protease cleavage site
▪ Promoter
▪ Signal peptides: phage protein translocation, crucial
▪ pIII protein is essential for infection of
bacteria
▪ Helper phage: wild-type pIII helper phage
and special helper phage
▪ Antigen immobilized on magnetic beads,
DNA recombination and gene manipulation; multiple cloning sites (MCS)
▪ Coat protein: PIII (larger protein, less than 5 copies,)
PVIII (more than 5 copies, decreased length)
Introduction of Phage Display Technology
The Ff bacteriophage structure
Introduction of Phage Display Technology
The scheme of phagemid vector
▪ IG region: intergenic region, usually contains the
polystryrene surfaces, or on columns, or is used in solution as biotinylated antigen and
Advantages of Phage Display for Recombinant Antibody Selection
Antibody Phage Display
Meiling Xiong 20180629
Contents
• Introduction of Ab phage Display Technology
• Ab Formats for Phage Display
• Ab Libraries Construction
• Producing the combinatorial library (ideally with 108 to 109 members) of
Antibody Formats
• The most commonly used format: single-chain variable fragment (scFv)
• More efficiently than through conventional hybridoma system.
• Cheaper to produce recombinant antibodies using bacteria, rather than mammalian cell line.
✓Simplicity of cloning process
✓Fast and easy library generation
✓A high display rate (small protein size ~25 kDa)
✓Less stable than Fab fragments
✓Tend to form dimers (can be reduced with linker more than 20 amino acids)
• Phage Ab Selection Methods & Strategies
• Phage Ab Screening Applications
• In vitro Affinity Maturation
• Expression & Purification of Phage Ab Fragments
Antibody Formats
• Fab
✓The light chain (VL-CL) and the Fddomain (VH-CH1) of the heavy chain of an antibody.
✓During bacterial expression, these two chains are synthesized separately, and secreted into the periplasm where they fold to form heterodimers.
often used for phage display, primarily the M13 and Fd strains.
▪ Proteins to be selected are infused to all
five coat proteins, with pIII and pVIII most commonly used.
packing sequence and replication origin of minus and plus strands
▪ Molecular tag: to facilitate library screening and for
protein analysis
▪ Restriction enul for
for display level
▪ Selective marker: for selection of infected host cells
Introduction of Phage Display Technology
▪ Nonlytic filamentous phage is the most
• Easier to maintain and grow bacterial cultures for recombinant antibody production.
• Bypass immunization in antibody selection.
• Bypass the use of animal cells for production of antibodies.