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PMID: 11433278 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter.

Nature biotechnology ·Vol. 19 ·No. 7 ·2001-07-00 ·Pages 656-60

Isalan M, Klug A, Choo Y

Abstract

DNA-binding domains with predetermined sequence specificity are engineered by selection of zinc finger modules using phage display, allowing the construction of customized transcription factors. Despite remarkable progress in this field, the available protein-engineering methods are deficient in many respects, thus hampering the applicability of the technique. Here we present a rapid and convenient method that can be used to design zinc finger proteins against a variety of DNA-binding sites. This is based on a pair of pre-made zinc finger phage-display libraries, which are used in parallel to select two DNA-binding domains each of which recognizes given 5 base pair sequences, and whose products are recombined to produce a single protein that recognizes a composite (9 base pair) site of predefined sequence. Engineering using this system can be completed in less than two weeks and yields proteins that bind sequence-specifically to DNA with Kd values in the nanomolar range. To illustrate the technique, we have selected seven different proteins to bind various regions of the human immunodeficiency virus 1 (HIV-1) promoter.

MeSH Terms
Amino Acid Sequence Base Sequence Binding, Competitive DNA/metabolism DNA-Binding Proteins/chemistry,metabolism Genetic Techniques HIV-1/genetics Kinetics Models, Molecular Molecular Sequence Data Peptide Library Peptides/chemistry Promoter Regions, Genetic Protein Binding Protein Structure, Tertiary Recombination, Genetic Sequence Homology, Amino Acid Sequence Homology, Nucleic Acid Zinc Fingers
Chemicals
DNA-Binding Proteins Peptide Library Peptides DNA
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Isalan M
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
Klug A
Choo Y
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30 references, click to expand
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Article Info
Journal
Nature biotechnology
Abbr.
Nat Biotechnol
ISSN
1087-0156
DOI
10.1038/90264
Published
2001-07-00
Pages
656-60
Language
English
Region
United States
NLM ID
9604648
PMCID
PMC2677679
Subset
IM
Grants
Medical Research Council · MC_U105184324 · United Kingdom
Medical Research Council · U.1051.04.011(78830) · United Kingdom
External Links
PubMed source DOI: 10.1038/90264 PMC Full Text View journal details
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