Covalent antibody display--an in vitro antibody-DNA library selection system.
The endonuclease P2A initiates the DNA replication of the bacteriophage P2 by making a covalent bond with its own phosphate backbone. This enzyme has now been exploited as a new in vitro display tool for antibody fragments. We have constructed genetic fusions of P2A with single-chain antibodies (scFvs). Linear DNA of these fusion proteins were processed in an in vitro coupled transcription-translation mixture of Escherichia coli S30 lysate. Complexes of scFv-P2A fusion proteins covalently bound to their own DNA were isolated after panning on immobilized antigen, and the enriched DNAs were recovered by PCR and prepared for the subsequent cycles of panning. We have demonstrated the enrichment of scFvs from spiked libraries and the specific selection of different anti-tetanus toxoid scFvs from a V-gene library with 50 million different members prepared from human lymphocytes. This covalent antibody display technology offers a complete in vitro selection system based exclusively on DNA-protein complexes.
Reiersen
Herald
H
Affitech AS, Oslo Research Park Gaustadalleen 21, N-0349 OSLO, Norway. herald@affitech.com
Løbersli
Inger
I
Løset
Geir Å
GÅ
Hvattum
Else
E
Simonsen
Bjørg
B
Stacy
John E
JE
McGregor
Duncan
D
Fitzgerald
Kevin
K
Welschof
Martin
M
Brekke
Ole H
OH
Marvik
Ole J
OJ
D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology; D017901:Bacteriophage P2 / Q000201:enzymology; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013745:Tetanus Toxoid / Q000276:immunology
D000914:Antibodies, Viral; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D013745:Tetanus Toxoid; D004706:Endodeoxyribonucleases