A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter.
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.
Isalan
M
M
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
Klug
A
A
Choo
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D005821:Genetic Techniques; D015497:HIV-1 / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D019151:Peptide Library; D010455:Peptides; D004247:DNA