Abstract
In this chapter we describe efficient procedures for construction, expression and screening of comprehensive libraries of murine 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 Plac. The L (or 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 P lac, Fd (composed of VH and CH1 domains) and x 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 pIII proteins. Enrichment of Fab phages with affinity for a specific antigen is then carried out by successive rounds of affinity purification using antigen-coated microtiter wells, immunotubes or plastic beads followed by reinfection of E. coli cells with the eluted bound phages (1-6). An outline of the method is illustrated in Figure 1.
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© 2001 Springer-Verlag Berlin Heidelberg
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Engberg, J., Jensen, L.B., Yenidunya, A.F., Brandt, K., Riise, E. (2001). Phage — Display Libraries of Murine Antibody Fab Fragments. In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Lab Manuals. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-04605-0_5
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DOI: https://doi.org/10.1007/978-3-662-04605-0_5
Publisher Name: Springer, Berlin, Heidelberg
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