RESUMEN
A library of long peptides displayed on the pIII protein of filamentous phage was used in biopanning experiments against several protein targets. We find that a large percentage of phage clones that bind specifically to a target contain peptide-encoding genes that do not have an ORF. Instead, the reading frame is either interrupted by one or more nonsuppressed stop codons, or a post-transcriptional frameshift is needed to account for the expression of the minor phage coat protein pIII. The percentage of frameshifted clones varies depending on the target. It can be as high as 90% for clones specific for soluble forms of certain cytokine receptors. Conversely, biopanning against four mAbs did not yield any frameshifted clones. Our studies focused on one clone that binds specifically to rat growth hormone binding protein (GHBP) yet does not have an ORF. A secondary peptide library containing random mutations of this sequence was constructed and panned against GHBP to optimize and correct the reading frame. In the last round (round two) of panning with this library, none of the phage clones that bound to GHBP had an ORF. However, careful analysis of these clones allowed us to design a synthetic peptide capable of binding to GHBP. The results of this study indicate that ORFs are not required to obtain gene expression of the minor coat protein of filamentous phage and suggest that some ORF- clones may have a selective advantage over the clones having ORFs.
Asunto(s)
Bacteriófagos/genética , Mutación del Sistema de Lectura/genética , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Técnicas Biosensibles , Cápside/genética , Proteínas Portadoras/genética , Clonación Molecular , Expresión Génica/genética , Hormona del Crecimiento/metabolismo , Datos de Secuencia Molecular , Oligopéptidos , Sistemas de Lectura Abierta/genética , Péptidos/química , Péptidos/inmunología , Unión Proteica/fisiología , Ratas , Análisis de Secuencia de ADNRESUMEN
Monoclonal antibody PAb1620 recognizes a conformational epitope on the transcription factor p53 and, upon binding, allosterically inhibits p53 binding to DNA. A highly diverse (1.5 x 10(10) members) phage-displayed library of peptides containing 40 random amino acids was used to identify the PAb1620 binding site on p53. Panning this library against PAb1620 resulted in three unique peptides which have statistically significant sequence identities with p53 sufficient to identify the binding site as being composed of amino acids 106-113 and 146-156. Based on these results, we propose a mechanism by which PAb1620 can allosterically inhibit p53 binding to DNA through an indirect interaction between the antibody binding site and the L1 loop (amino acids 112-124) of p53, which is a component of the DNA binding region.