title: Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection.
abstract: Engineered Cys2His2 zinc finger proteins (ZFPs) can mediate regulation of endogenous gene expression in mammalian cells. Ideally, all zinc fingers in an engineered multifinger protein should be optimized concurrently because cooperative and context-dependent contacts can affect DNA recognition. However, the simultaneous selection of key contacts in even three fingers from fully randomized libraries would require the consideration of >10(24) possible combinations. To address this challenge, we have developed a novel strategy that utilizes directed domain shuffling and rapid cell-based selections. Unlike previously described methods, our strategy is amenable to scale-up and does not sacrifice combinatorial diversity. Using this approach, we have successfully isolated multifinger proteins with improved in vitro and in vivo function. Our results demonstrate that both DNA binding affinity and specificity are important for cellular function and also provide a general approach for optimizing multidomain proteins.
authors.affiliation: Molecular Pathology Unit, Division of Molecular Pathology and Research, Department of Pathology, Massachusetts General Hospital, Charlestown, MA 02129, USA.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Hurt
authors.forename: Stacey A
authors.initials: SA
authors.lastname: Thibodeau
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Hirsh
authors.forename: Carl O
authors.initials: CO
authors.lastname: Pabo
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016044:Fusion Proteins, bcr-abl / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006678:HIV / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D019151:Peptide Library; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D016044:Fusion Proteins, bcr-abl; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins