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

Nature Biotechnology volume 19, pages 656–660 (2001)Cite this article

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.

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Figure 1: Overview of the protein engineering strategy.
Figure 2: Composition of the “bipartite” library.
Figure 3: Matrix specificity assay for seven zinc finger DNA-binding domains designed to bind sequences in the HIV-1 promoter.
Figure 4: Gel shift assays to determine the apparent equilibrium dissociation constants (Kd values) of three engineered zinc finger peptides.
Figure 5: Matrix sequence-discrimination assay for three-zinc finger peptides generated using three regions of the HIV-1 LTR.
Figure 6: Construction of a gene cassette coding for the zinc finger phage-display libraries used in this study.

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Acknowledgements

This work was supported by the Medical Research Council, UK..

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  1. Mark Isalan & Yen Choo

    Present address: Gendaq Ltd, 1-3 Burtonhole Lane, London, NW7 1AD, UK

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  1. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    Mark Isalan, Aaron Klug & Yen Choo

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Correspondence to Yen Choo.

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Isalan, M., Klug, A. & Choo, Y. A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter. Nat Biotechnol 19, 656–660 (2001). https://doi.org/10.1038/90264

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