Cell-free selection of zinc finger DNA-binding proteins using in vitro compartmentalization.
We have exploited emulsion-based in vitro compartmentalization (IVC) to devise a method for the selection of zinc finger proteins (ZFPs) on the basis of their DNA-binding specificity. A library of ZFPs fused to a C-terminal peptide tag is encoded by a set of DNA cassettes that are prepared wholly in vitro. In addition to the ZFP gene, each DNA cassette also carries a given DNA target binding site sequence for which one wishes to isolate ZFP binders. An aliquot of the library is added to bacterial S30 extract and emulsified in mineral oil so that most of the aqueous droplets contain, on average, no more than one gene. If an intra-compartmentally expressed ZFP binds specifically to its encoding DNA via the target binding site, the complex can be purified by affinity capture via the peptide tag after breaking the emulsion, thus rescuing the gene. We present proof-of-principle for this IVC selection method by selecting a specific high-affinity ZFP gene from a high background of a related gene. We also propose that high-affinity ZFPs can be used as genotype-phenotype linkages to enable selection of other proteins using IVC.
Sepp
Armin
A
Sangamo BioSciences Inc. Point Richmond Tech Center II, Richmond, CA 94804, USA.
Choo
Yen
Y
D001665:Binding Sites; D002451:Cell Compartmentation; D002474:Cell-Free System / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D019151:Peptide Library; D014158:Transcription, Genetic; D016335:Zinc Fingers
D017931:DNA Primers; D004268:DNA-Binding Proteins; D019151:Peptide Library