Phage Display Technology
Over the past few years, considerable effort has gone into genetic engineering and manipulation of antibody molecules. One consequence of this research has been the use of filamentous phage as a vehicle on which to display antibody fragments. It is possib
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37 Phage Display Technology Protocols Michael Johns and Donald B. Palmer 1. Introduction Over the past few years, considerable effort has gone into genetic engineering and manipulation of antibody molecules. One consequence of this research has been the use of filamentous phage as a vehicle on which to display antibody fragments. It is possible to express a variety of molecules on the surface of phage, including peptides and antibody fragments. With regard to antibodies, libraries of both sFv and Fab fragments have been successfully cloned onto the surface of bacteriophages, usually via the N terminal of the gene III product, although expression on the gene VIII product has also been employed to isolate low-affinity antibodies. A variety of different libraries of phage expressing randomly combined VH and VL chain genes have been created from different sources, e.g., mouse, human, and chicken. One of the most commonly used libraries is that of Nissim et al. (1), who has developed a semisynthetic phage library of >108 different specificities. The library has been made in vitro from 50 cloned human VH gene segments, which have random nucleotide sequences encoding CDR3 lengths of 4–12 residues, combined with a single VL. They have used the library to select a variety of sFv against 18 different antigens after four or five rounds of panning. The selected phages and sFv were used in western blotting and also for epitope mapping and staining of cells. De Kruif et al. (2) have also constructed a semisynthetic sFv library using cloned human germline VH genes and synthetic CDR3. However, in contrast to Nissim et al., they have used a 6–15 amino-acid CDR3 containing short From: Methods in Molecular Medicine, Vol. 40: Diagnostic and Therapeutic Antibodies Edited by: A. J. T. George and C. E. Urch © Humana Press Inc., Totowa, NJ
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stretches of fully randomized amino acid residues flanked by regions of limited variability (selected on the frequency of amino acids in the CDR3 of natural antibodies). In doing so the authors reason that this will increase occurrence of phage displaying functionally useful fragments. They also attempted to increase variability by using seven different light chains from both h and g subclasses rather than h3 alone, as Nissim et al. (1) used. Griffiths et al. (3) have produced a Fab library of 6.5 × 1010 specificities using a combinatorial infection process that involves transforming bacteria with a library of heavy chains on a plasmid and then infecting the culture with a library of light chains on a phage and also a Cre recombinase on a separate phage. The Cre recombinase then uses lox P sites within the library vectors to randomly combine H and L chains (from the plasmid and phage vectors) on the same phage replicon within each bacterium. With the increased size of this library, the authors report antibody fragments of affinities >80-fold higher than those isolated from smaller libraries. A similar library in the sFv format has also been developed from the same VH a
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