In vivo biotinylated proteins as targets for phage-display selection experiments

Screening phage-displayed combinatorial libraries represents an attractive method for identifying affinity reagents to target proteins

Michael D Scholle; Frank R Collart; Brian K Kay

2004

Scholarcy highlights

  • Screening phage-displayed combinatorial libraries represents an attractive method for identifying affinity reagents to target proteins
  • Two critical components of a successful selection experiment are having a pure target protein and its immobilization in a native conformation. To achieve both of these requirements in a single step, we have devised cytoplasmic expression vectors for expression of proteins that are tagged at the amino- or carboxy-terminus via the AviTag, which is biotinylated in vivo with concurrent expression of the BirA biotin ligase
  • To facilitate implementation in high-throughput applications, the engineered vectors, pMCSG15 and pMCSG16, contain a ligase-independent cloning site, which permits up to 100% cloning efficiency
  • The expressed protein can be purified from bacterial cell lysates with immobilized metal affinity chromatography or streptavidin-coated magnetic beads, and the beads used directly to select phage from combinatorial libraries
  • To demonstrate the utility of pMCSG16, a set of 192 open reading frames were cloned, and protein was expressed and immobilized for use in high-throughput selections of phage-display libraries

Need more features? Save interactive summary cards to your Scholarcy Library.