Assuntos
Algoritmos , Bioensaio/métodos , Técnicas de Cultura de Células/métodos , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Robótica/métodos , beta-Galactosidase/metabolismo , Bioensaio/instrumentação , Técnicas de Cultura de Células/instrumentação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Reporter/genética , Reprodutibilidade dos Testes , Robótica/instrumentação , Sensibilidade e Especificidade , beta-Galactosidase/genéticaRESUMO
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.
Assuntos
Dedos de Zinco/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , DNA Recombinante/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fusão bcr-abl/química , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , HIV/genética , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Biblioteca de Peptídeos , Engenharia de Proteínas , Estrutura Terciária de Proteína , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Seleção Genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Extract: The Cys2His2 zinc finger domain (hereafter simply "zinc finger") provides a useful scaffold for creating customized DNA-binding proteins, a technology with potential applications in biological research, molecular medicine, and gene therapy. Using a combination of targeted randomization and selection methodologies (e.g., phage display), many research groups have successfully altered the DNA-binding specificities of single zinc fingers, which typically recognize three to four base pairs of DNA. In these experiments, potential DNA-binding residues in a finger's a-helix (or "recognition helix") were randomized to generate a library of variants and then selection methods were used to identify fingers with desired DNA-binding specificities. To create synthetic multi-finger proteins capable of recognizing longer DNA sequences, various investigators have linked together three or more pre-selected or pre-characterized finger domains. This type of strategy, which assumes that individual fingers behave in a modular fashion, permits the rapid assembly of multi-finger proteins directed to bind a wide variety of different DNA sequences.
