[5] Screening of phage antibody libraries

Publisher Summary A variety of vectors have been used for the presentation of antibodies on the phage surface. These include phage vectors that encode the pill fusion and all the functions required for replication, packaging, and infection of bacteria and phagemid vectors that require “rescue” with a helper phage. Phagemid vectors comprise the pill fusion, plasmid, and phage origins of replication and antibiotic resistance markers; the helper phage provides the other functions for single-stranded replication and packaging. Helper phages are poorly packaged, in competition with phagemids, due to a defective origin (Vieira and Messing). This chapter describes the protocols suitable for helper VCS M13 (Stratagene, La Jolla, CA) and M13 K07, both of which confer kanamycin resistance on their hosts. Because the phagemid pIII fusion competes with helper phage pill for incorporation into the phage coat, majority of the phage particles display none or only one antibody molecule. Proteolytic cleavage of antibody, from the surface of phage, also reduces the number of antibody fragments displayed.