Abstract
In this chapter we describe efficient procedures for the construction, expression and screening of comprehensive libraries of human Fab antibody fragments displayed on the surface of filamentous phage. Phagemid vectors are used for placing randomly paired light (L) and heavy (H) chain coding regions under transcriptional control of Plac. The H chain coding region is fused in-frame with the phage gene, ΔgIII, coding for a truncated version of the phage surface protein pIII (ΔpIII). After superinfection with helper phage and induction of Plac, Fd (composed of VH and CH1 domains) and κ-or α-L chains assemble into Fab fragments in the periplasm of the Escherichia coli host strain, and the Fab-ΔpIII protein complex is displayed at one end of the phage by displacing one (or more) of the wild-type pIII proteins. Enrichment of Fab phages with affinity for a selected “antigen” is then carried out by successive rounds of affinity purification using “antigen”-coated immunotubes or plastic beads followed by reinfection of E. coli cells with the eluted bound phages (see refs. 1–10). An outline of the method is illustrated in Fig. 1.
Overview of the steps involved in the generation of human antibody Fab libraries. Messenger RNA is isolated from peripheral blood of donors and copied into cDNA. This material is then used as template for PCR amplification of immunoglobulin gene fragments, which in turn are cloned and expressed as functional Fab molecules in fusion with a phage surface protein (pIII) as mentioned in the text. The entire mixture of phage-displayed Fab molecules are then submitted to affinity purification methods to obtain specific binders to a selected “antigen.”
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Engberg, J. et al. (2003). Human Recombinant Fab Antibodies with T-Cell Receptor-Like Specificities Generated from Phage Display Libraries. In: Welschof, M., Krauss, J. (eds) Recombinant Antibodies for Cancer Therapy. Methods in Molecular Biology™, vol 207. Humana Press. https://doi.org/10.1385/1-59259-334-8:161
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DOI: https://doi.org/10.1385/1-59259-334-8:161
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