RESUMEN
Randomized ZFP-TF libraries could induce a specific phenotype without detailed knowledge about the phenotype of interest because, theoretically, the libraries could modulate any gene in the target organism. We have developed a novel method for enhancing the efficiency of recombinant protein production in mammalian and microbial cells using combinatorial libraries of zinc finger protein transcription factors. To this end, we constructed tens of thousands of zinc finger proteins (ZFPs) with distinct DNA-binding specificities and fused these ZFPs to either a transcriptional activation or repression domain to make transcriptional activators or repressors, respectively. Expression vectors that encode these artificial transcription factors were delivered into Saccharomyces cerevisiae or HEK 293 cells along with reporter plasmids that code for human growth hormone (hGH) or SEAP (secreted alkaline phosphatase) (for yeast or HEK, respectively). Expression of the reporter genes was driven by either the cytomegalovirus (CMV) or SV40 virus promoters. After transfection, we screened the cells for increased synthesis of the reporter proteins. From these cells, we then isolated several ZFP-transcription factors (ZFP-TFs) that significantly increased hGH or SEAP synthesis and subjected these regulatory proteins to further characterization. Our results show that randomized ZFP-TF libraries are useful tools for improving the yield of heterologous recombinant protein both in yeast and mammalian cells.
Asunto(s)
Fosfatasa Alcalina/biosíntesis , Hormona de Crecimiento Humana/biosíntesis , Riñón/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc/genética , Fosfatasa Alcalina/genética , Línea Celular , Mejoramiento Genético/métodos , Hormona de Crecimiento Humana/genética , Humanos , Biblioteca de Péptidos , Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
We have developed a method in which randomized libraries of zinc finger-containing artificial transcription factors are used to induce phenotypic variations in yeast and mammalian cells. By linking multiple zinc-finger domains together, we constructed more than 100,000 zinc-finger proteins with diverse DNA-binding specificities and fused each of them to either a transcription activation or repression domain. The resulting transcriptional regulatory proteins were expressed individually in cells, and the transfected cells were screened for various phenotypic changes, such as drug resistance, thermotolerance or osmotolerance in yeast, and differentiation in mammalian cells. Genes associated with the selected phenotypes were also identified. Our results show that randomized libraries of artificial transcription factors are useful tools for functional genomics and phenotypic engineering.
Asunto(s)
Clonación Molecular/métodos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Biblioteca de Péptidos , Fenotipo , Ingeniería de Proteínas/métodos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de Zinc/genética , Animales , Células Cultivadas , Células Eucariotas/metabolismo , Regulación de la Expresión Génica/genética , Variación Genética , Ratones , Mutagénesis Sitio-Dirigida/genética , Mioblastos/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
We describe methods for generating artificial transcription factors capable of up- or downregulating the expression of genes whose promoter regions contain the target DNA sequences. To accomplish this, we screened zinc fingers derived from sequences in the human genome and isolated 56 zinc fingers with diverse DNA-binding specificities. We used these zinc fingers as modular building blocks in the construction of novel, sequence-specific DNA-binding proteins. Fusion of these zinc-finger proteins with either a transcriptional activation or repression domain yielded potent transcriptional activators or repressors, respectively. These results show that the human genome encodes zinc fingers with diverse DNA-binding specificities and that these domains can be used to design sequence-specific DNA-binding proteins and artificial transcription factors.