Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 100, Issue 21, Pages 12271-12276Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2135381100
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Funding
- NCI NIH HHS [T32 CA009216, 5T32 CA 09216] Funding Source: Medline
- NIDDK NIH HHS [K08 DK002883, K08 DK 02883] Funding Source: Medline
- NIH HHS [DP1 OD006862] Funding Source: Medline
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Engineered Cys(2)His(2) 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.
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