Simplified gene-fragment phage display system for epitope mapping.
We describe a simple and efficient system for epitope mapping by cloning random gene fragments into a specially designed gIIIp-based phage display vector. DNA encoding the antigen of interest is PCR-amplified and partially digested 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 gIII after linearizing the vector with SmaI/SrfI, and the ligation is carried out in the presence of restriction enzyme SrfI. The restriction enzyme in the ligation reaction recuts the self-ligated vector but not the recombinants, since ligation with foreign fragments destroys the enzyme recognition site. Dephosphorylation of inserts prevents their chimerization and ensures ligation of single insert per vector molecule. Thus, using 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 compared 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 transformants. This step eliminates all the phages that carry insert that are not in-frame with gIIIp and therefore do not display gIIIp. We have shown the utility of the above system in constructing a glutathione-S-transferase (GST) gene-fragment library in phages and identifying the epitope recognized by a monoclonal antibody against GST.
Gupta
S
S
University of Delhi South Campus, India.
Arora
K
K
Sampath
A
A
Khurana
S
S
Singh
S S
SS
Gupta
A
A
Chaudhary
V K
VK
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018604:Epitope Mapping / Q000379:methods*; D004926:Escherichia coli / Q000821:virology; D015723:Gene Library; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012549:Schistosoma japonicum / Q000201:enzymology
D000911:Antibodies, Monoclonal; D005982:Glutathione Transferase