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PEDS Advance Access published online on September 26, 2005
Protein Engineering Design and Selection, 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
Received May 31, 2005
Revised August 8, 2005
Accepted August 8, 2005

Article

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

Kristina Jonas 1 3, Erhard Van Der Vries 2 3, Mikael T. I. Nilsson 3, and Mikael Widersten 4*

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

* To whom correspondence should be addressed.
Mikael Widersten, E-mail: mikael.widersten{at}biokemi.uu.se


  Abstract

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.
3These authors contributed equally to this work
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