Repertoire selection of variant single-chain Cro: toward directed DNA-binding specificity of helix-turn-helix proteins

Biochemistry
Mikael T I Nilsson, M Widersten

Abstract

A single-chain derivative of the lambda Cro repressor (scCro) has been randomly mutated in amino acid residues critical for specific DNA recognition to create libraries of protein variants. Utilizing phage display-afforded affinity selection, scCro variants have been isolated for binding to synthetic DNA ligands. Isolated scCro variants were analyzed functionally, both in fusion with phage particles and after expression of the corresponding free proteins. The binding properties with regard to specificity and affinity in binding to different DNA ligands were investigated by inhibition studies and determination of equilibrium dissociation constants for formed complexes. Variant proteins with altered DNA-sequence specificity were identified, which favored binding of targeted synthetic DNA sequences over a consensus operator sequence, bound with high affinity by wild-type Cro. The specificities were relatively modest (2-3-fold, as calculated from K(D) values), which can be attributed to the inherent properties in the design of the selection system; one half-site of the synthetic DNA sequences maintains the consensus operator sequence, and one "subunit" of the variant single-chain Cro dimers was conserved as wild-type sequence. The ...Continue Reading

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Citations

Sep 25, 2010·Briefings in Functional Genomics·Marcel Geertz, Sebastian J Maerkl
Oct 15, 2005·Nucleic Acids Research·Atanu Maiti, Siddhartha Roy
Apr 7, 2007·Nucleic Acids Research·Petri Henriksson-PeltolaElisabeth Haggård-Ljungquist
Nov 3, 2015·Journal of Bioscience and Bioengineering·Takaaki KojimaHideo Nakano
Aug 2, 2018·Bioscience, Biotechnology, and Biochemistry·Takaaki KojimaHideo Nakano
May 24, 2019·Bioscience, Biotechnology, and Biochemistry·Takaaki KojimaHideo Nakano

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