Phage-Display Libraries of Murine and Human Antibody Fab Fragments
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This chapter describes efficient procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab 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 P lac . The L (or H) chain-coding region is fused in-frame with the truncated phage gene, ΔgIII, coding for a truncated version of the phage surface protein pill (ΔpIII). After superinfection with helper phage and induction of P lac , Fd (composed of VH and CH1 domains), and κ (or λ) L chains assemble into Fab fragments in the periplasm, and the Fab-ΔpIII protein complex is displayed at one end of the phage by displacing one (or more) of the wild-type pill proteins. Enrichment of Fab phages with affinity for a specific antigen is then done by successive rounds of affinity purification in antigen-coated microtiter wells or immunotubes and reinfection of Escherichia coli cells by the eluted bound phages (1, 2, 3, 4, 5, 6).
KeywordsPolymerase Chain Reaction Program Polymerase Chain Reaction Tube Variable Heavy Link Fragment pelB Leader
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