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A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins

Authors
  • Speight, R.E.
  • Hart, D.J.
  • Sutherland, J.D.
  • Blackburn, J.M.
Publication Date
Jan 01, 2001
Source
Queensland University of Technology ePrints Archive
Keywords
License
Unknown

Abstract

Background Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor κB (NF-κB) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene. Results A range of model fusion proteins to either the amino- or carboxy-terminus of NF-κB p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-κB p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:108 and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections. Conclusions A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

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