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PEDS Advance Access originally published online on September 26, 2005
Protein Engineering Design and Selection 2005 18(11):537-546; doi:10.1093/protein/gzi058
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Isolation of novel single-chain Cro proteins targeted for binding to the bcl-2 transcription initiation site by repertoire selection and subunit combinatorics

Kristina Jonas1,2,3, Erhard Van Der Vries1,3,4, Mikael T.I. Nilsson1,5 and Mikael Widersten1,6

1Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden 2Present address: Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden 4Present address: Developmental Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands 5Present address: BioDesign Institute–BON Center, Main Arizona State University, 1001 S. McAllister Avenue, Tempe, AZ 85287-5201, USA

6 To whom correspondence should be addressed. E-mail: mikael.widersten{at}biokemi.uu.se

New designed DNA-binding proteins may be recruited to act as transcriptional regulators and could provide new therapeutic agents in the treatment of genetic disorders such as cancer. We have isolated tailored DNA-binding proteins selected for affinity to a region spanning the transcription initiation site of the human bcl-2 gene. The proteins were derived from a single-chain derivative of the lambda Cro protein (scCro), randomly mutated in its recognition helices to construct libraries of protein variants of distinct DNA-binding properties. By phage display-afforded affinity selections combined with recombination of shuffled subunits, protein variants were isolated, which displayed high affinity for the target bcl-2 sequence, as determined by electrophoretic mobility shift and biosensor assays. The proteins analyzed were moderately sequence-specific but provide a starting point for further maturation of desired function.

Keywords: phage display/protein–DNA interactions/repressor/single-chain Cro/subunit shuffling

Received May 31, 2005; revised August 8, 2005; accepted August 8, 2005.

Edited by Brian Matthews


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