Towards the design of an antibody that recognises a given protein epitope.
We have explored the possibility of designing repertoires of antibodies complementary to a given protein epitope, specifically the face of the ribonuclease inhibitor barstar that binds to the enzyme barnase. An antibody repertoire was created by mutation of ten residues in the hypervariable loops of a synthetic antibody fragment and displayed on filamentous bacteriophage. The positions of three of the ten residues of the antibody (VL 32, 50 and 94) were chosen to match a triangle of three negative charges on the face of barstar and mutated to favour residues of opposite charge or those with hydrogen-bonding potential. The other seven residues, chosen to allow for variation in the surface of interaction, were mutated at random. One of the antibody fragments isolated after selection of the repertoire (10(8) clones per library) was shown to bind to barstar with an affinity of 1.0x10(-7) M and the binding was competed by barnase. Furthermore, the binding of the antibody to barstar was highly sensitive to mutation of any of five residues of barstar known to contact barnase. This indicates that it may be possible, by a combination of design and selection, to build antibodies to a given epitope.
Kirkham
P M
PM
MRC Centre, Cambridge Centre for Protein Engineering, Hills Road, Cambridge, CB2 2QH, UK.
Neri
D
D
Winter
G
G
D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics; D001665:Binding Sites / Q000276:immunology; D001667:Binding, Competitive / Q000276:immunology; D003001:Cloning, Molecular; D000939:Epitopes / Q000276:immunology*; D008958:Models, Molecular; D009154:Mutation; D009994:Osmolar Concentration; D011485:Protein Binding / Q000276:immunology; D015202:Protein Engineering / Q000379:methods*; D012260:Ribonucleases / Q000276:immunology
D000906:Antibodies; D001426:Bacterial Proteins; D000939:Epitopes; C057438:barstar protein, Bacillus amyloliquefaciens; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease