RESUMEN
We have successfully linked protein library screening directly with the identification of active proteins, without the need for individual purification, display technologies or physical linkage between the protein and its encoding sequence. By using 'MAX' randomization we have rapidly constructed 60 overlapping gene libraries that encode zinc finger proteins, randomized variously at the three principal DNA-contacting residues. Expression and screening of the libraries against five possible target DNA sequences generated data points covering a potential 40,000 individual interactions. Comparative analysis of the resulting data enabled direct identification of active proteins. Accuracy of this library analysis methodology was confirmed by both in vitro and in vivo analyses of identified proteins to yield novel zinc finger proteins that bind to their target sequences with high affinity, as indicated by low nanomolar apparent dissociation constants.
Asunto(s)
Técnicas Químicas Combinatorias , Proteínas de Unión al ADN/genética , Biblioteca de Genes , Dedos de Zinc , Sitios de Unión , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Biblioteca de Péptidos , Proteínas/química , Proteínas/genética , Proteínas/aislamiento & purificación , Análisis de Secuencia de Proteína , Técnicas del Sistema de Dos Híbridos , Levaduras/genéticaRESUMEN
A simple protein-DNA interaction analysis has been developed using a high-affinity/high-specificity zinc finger protein. In essence, purified protein samples are immobilized directly onto the surface of microplate wells, and fluorescently labeled DNA is added in solution. After incubation and washing, bound DNA is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.2 nM DNA. Since the detection of bound DNA is noninvasive and the protein-DNA interaction is not disrupted during detection, iterative readings may be taken from the same well, after successive alterations in interaction conditions, if required. In this respect, the assay may therefore be considered real time and permits appropriate interaction conditions to be determined quantitatively. The assay format is ideally suited to investigate the interactions of purified unlabeled DNA binding proteins in a high-throughput format.
Asunto(s)
Técnicas Biosensibles/métodos , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/química , ADN/análisis , ADN/química , Mapeo de Interacción de Proteínas/métodos , Espectrometría de Fluorescencia/métodos , Adsorción , Sitios de Unión , Técnicas Biosensibles/instrumentación , Análisis de Falla de Equipo , Unión Proteica , Mapeo de Interacción de Proteínas/instrumentación , Espectrometría de Fluorescencia/instrumentación , Dedos de ZincRESUMEN
A simple protein-DNA interaction analysis has been developed using both a high-affinity/high-specificity zinc finger protein and a low-specificity zinc finger protein with nonspecific DNA binding capability. The latter protein is designed to mimic background binding by proteins generated in randomized or shuffled gene libraries. In essence, DNA is immobilized onto the surface of microplate wells via streptavidin capture, and green fluorescent protein (GFP)-labeled protein is added in solution as part of a crude cell lysate or protein mixture. After incubation and washing, bound protein is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.4 nM protein. The assay format is ideally suited to investigate the interactions of DNA binding proteins from within crude cell extracts and/or mixtures of proteins that may be encountered in protein libraries generated by codon randomization or gene shuffling.