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Protein Engineering, Vol. 13, No. 7, 519-526, July 2000
© 2000 Oxford University Press

Functional expression and affinity selection of single-chain Cro by phage display: isolation of novel DNA-binding proteins

Mikael T.I. Nilsson1, Michael C. Mossing2 and Mikael Widersten1,3

1 Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden and 2 Department of Chemistry, University of Mississippi, University, MS 38677, USA

A robust selection system affording phage display of the DNA-binding helix–turn–helix protein Cro is presented. The aim of the work was to construct an experimental system allowing for the construction and isolation of Cro-derived protein with new DNA-binding properties. A derivative of the phage {lambda} Cro repressor, scCro8, in which the protein subunits had been covalently connected via a peptide linker was expressed in fusion with the gene 3 protein of Escherichia coli filamentous phage. The phage-displayed single-chain Cro was shown to retain the DNA binding properties of its wild-type Cro counterpart regarding DNA sequence specificity and binding affinity. A kinetic analysis revealed the rate constant of dissociation of the single-chain Cro-phage/DNA complex to be indistinguishable from that of the free single-chain Cro. Affinity selection using a biotinylated DNA with a target consensus operator sequence allowed for a 3000-fold enrichment of phages displaying single-chain Cro over control phages. The selection was based on entrapment of phage/DNA complexes formed in solution on streptavidin-coated paramagnetic beads. The expression system was subsequently used to isolate variant scCro8 proteins, mutated in their DNA-binding residues, that specifically recognized new, unnatural target DNA ligands.


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