RESUMEN
Engineered zinc-finger transcription factors (ZF-TF) are powerful tools to modulate the expression of specific genes. Complex libraries of ZF-TF can be delivered into cells to scan the genome for genes responsible for a particular phenotype or to select the most effective ZF-TF to regulate an individual gene. In both cases, the construction of highly representative and unbiased libraries is critical. In this protocol, we describe a user-friendly ZF technology suitable for the creation of complex libraries and the construction of customized ZF-TFs. The new technology described here simplifies the building of ZF libraries, avoids PCR-introduced bias and ensures equal representation of every module. We also describe the construction of a customized ZF-TF that can be transferred to a number of expression vectors. This protocol can be completed in 9-11 d.
Asunto(s)
Biblioteca de Péptidos , Ingeniería de Proteínas/métodos , Dedos de Zinc/genética , Animales , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Vectores Genéticos , Humanos , Plásmidos/genética , Proteínas Recombinantes/genética , Factores de Transcripción/genéticaRESUMEN
Phage-displayed peptides that selectively bind to aldolase catalytic antibody 93F3 when bound to a particular 1,3-diketone hapten derivative have been developed using designed selection strategies with libraries containing 7-12 randomized amino acid residues. These phage-displayed peptides discriminated the particular 93F3-diketone complex from ligand-free 93F3 and from 93F3 bound to other 1,3-diketone hapten derivatives. By altering the selection procedures, phage-displayed peptides that bind to antibody 93F3 in the absence of 1,3-diketone hapten derivatives have also been developed. With using these phage-displayed peptides, ligand-bound states of the antibody were distinguished from each other. A docking model of one of the peptides bound to the antibody 93F3-diketone complex was created using a sequential divide-and-conquer peptide docking strategy; the model suggests that the peptide interacts with both the antibody and the ligand through a delicate hydrogen bonding network.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Bacteriófago M13/metabolismo , Sitios de Unión de Anticuerpos , Catalasa/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Animales , Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/genética , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Bacteriófago M13/química , Catalasa/inmunología , Humanos , Cetonas/química , Cetonas/metabolismo , Ligandos , Ratones , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Oligopéptidos/inmunología , Biblioteca de PéptidosRESUMEN
A 21-mer peptide that can be used to covalently introduce synthetic molecules into proteins has been developed. Phage-displayed peptide libraries were subjected to reaction-based selection with 1,3-diketones. The peptide was further evolved by addition of a randomized region and reselection for improved binding. The resulting 21-mer peptide had a reactive amino group that formed an enaminone with 1,3-diketone and was used as a tag for labeling of maltose binding protein. Using this peptide tag and 1,3-diketone derivatives, a variety of molecules such as reporter probes and functionalities may be covalently introduced into proteins of interest.