Simplified gene-fragment phage display system for epitope mapping

We describe a simple and efficient system for epitope mapping by cloning ra ndom gene fragments into a specially designed gIIIp-based phage display vec tor: DNA encoding the antigen of interest is PCR-amplified and partially di gested with DNaseI to generate 50-150-bp-long fragments, which are polished with T4 DNA polymerase and dephosphorylated. These fragments are cloned at the 5' end of the gill after linearizing the vector with SmaI/SrfI, and th e ligation is carried out in the presence of restriction enzyme SN. The res triction enzyme in the ligation reaction recurs the self-ligated vector but not the recombinants, since ligation with foreign fragments destroys the e nzyme recognition site. Dephosphorylation of inserts prevents their chimeri zation and ensures ligation of single insert per vector molecule. Thus, usi ng the above strategy, which prevents self-ligation of both the insert and the vector the overall cloning efficiency and, thereby the library size, is improved more than 10-fold com- pared to the standard blunt-end, ligation- based methods for making similar libraries. The library is further enriched by a single-step infection of E. coli by phages obtained from primary tran sformants. This step eliminates all the phages that carry insert that are n ot in-frame with gIIIp and therefore do (B) not display gIIIp. We have show n the utility of the above system in constructing a glutathione-S-transfera se (GST) gene-fragment library in phages and identifying the epitope recogn ized by a monoclonal antibody against GST.