Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains.
The display of proteins on the surface of phage offers a powerful means of selecting for rare genes encoding proteins with binding activities. Recently we found that antibody heavy and light chain variable (V) domains fused as a single polypeptide chain to a minor coat protein of filamentous phage fd, could be enriched by successive rounds of phage growth and panning with antigen. This allows the selection of antigen-binding domains directly from diverse libraries of V-genes. Now we show that heterodimeric Fab fragments can be assembled on the surface of the phage by linking one chain to the phage coat protein, and secreting the other into the bacterial periplasm. Furthermore by introducing an amber mutation between the antibody chain and the coat protein, we can either display the antibody on phage using supE strains of bacteria, or produce soluble Fab fragment using non-suppressor strains. The use of Fab fragments may offer advantages over single chain Fv fragments for construction of combinatorial libraries.
Hoogenboom
H R
HR
Centre for Protein Engineering, Cambridge, UK.
Griffiths
A D
AD
Johnson
K S
KS
Chiswell
D J
DJ
Hudson
P
P
Winter
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002213:Capsid / Q000096:biosynthesis; D003090:Coliphages / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis*; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis*; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000096:biosynthesis; D011995:Recombination, Genetic; D014760:Viral Fusion Proteins / Q000096:biosynthesis*
D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D021382:Protein Sorting Signals; D014760:Viral Fusion Proteins