Functional expression and affinity selection of single-chain Cro by phage display: isolation of novel DNA-binding proteins
2000 (English)In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, ISSN 0269-2139, Vol. 13, no 7, 519-526 p.Article in journal (Refereed) Published
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 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.
Place, publisher, year, edition, pages
2000. Vol. 13, no 7, 519-526 p.
Biochemistry and Molecular Biology
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:uu:diva-37991OAI: oai:DiVA.org:uu-37991DiVA: diva2:65890