A Novel Helper Phage Enabling Construction of Genome-Scale ORF-Enriched Phage Display Libraries

We describe the design of a novel helper phage, AGM13, which carries two copies of trypsin cleavage sites in the linker domains of gIIIP

Amita Gupta; Nimisha Shrivastava; Payal Grover; Ajay Singh; Kapil Mathur; Vaishali Verma; Charanpreet Kaur; Vijay K. Chaudhary

2013

Scholarcy highlights

  • Phage display is a powerful technique for studying proteinligand interactions and identification of immunodominant regions using gene fragment libraries
  • AGM13 phages were highly sensitive to trypsin; treatment with 0.1 mg/ml trypsin reduced the titre of infectious phages by 6 orders of magnitude, and by an additional 3 orders of magnitude at 10 mg/ml, indicating that the novel trypsin cleavage sites in gIIIP were fully exposed
  • We designed a novel helper phage, AGM13, which was employed to produce genome-scale open reading frame-enriched libraries of gene/genome fragments cloned into a gIIIP-based phage display vector
  • There is a trypsin site located just before the first codon of wild-type gIIIP in the phagemid vector, which results in the conversion of phagemid encoded trypsin-resistant gIIIP fusion protein into trypsin-resistant gIIIP with full infectivity upon trypsin treatment
  • T7 phage has been successfully employed for ORF selection in large libraries to obtain up to 90% ORF enrichment
  • Upon rescue of the phagemid-borne gene fragment library with AGM13, the in-frame clones encode trypsin-resistant gIIIP fusion protein, which becomes incorporated into the phages, along with other copies of helper phage AGM13encoded trypsin-sensitive gIIIP
  • AGM13 enables the high efficiency selection of reading frames and AGM13 has a wide range of applications in the production of open reading frame-enriched phage displayed cDNA, gene-fragment and antibody libraries

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