Functional protein microarrays for parallel characterisation of p53 mutants

Abstract

Understanding the way in which single nucleotide polymorphisms and mutations in the human genome result in individual susceptibility to disease is a major goal in the postgenomic era. Such knowledge should accelerate the development of personalised medicine in which drug treatment can specifically match an individual's genotype. High-throughput DNA sequencing is generating the initial information required, but new technologies are required that can rapidly characterise the phenotypic effects of the identified polymorphisms. For example, many thousands of allelic variants of the p53 gene have been described and are responsible for more than 50% of cancers, however few of the protein products have been functionally characterised. Here we have quantified in parallel the effects of mutations and polymorphisms on the DNA-binding function of the p53 oncoprotein using a protein microarray, allowing their subclassification according to functional effect. Protein-protein interactions between p53 variants and (i) a regulatory oncoprotein, (ii) a regulatory kinase resulting in on-chip phosphorylation, are also described, suggesting the more general utility of this high-throughput assay format.

A Simple Parallel Analytical Method of Prenatal Screening

“…The advance in protein microarray technology has benefited the medical community greatly, making the parallel analysis of multiple correlative biomarkers a reality [1,2] . In the past few years, researchers all over the world have developed a number of protein microarray products for use in diagnosis, prognosis, prevention and therapy [3][4][5][6][7][8] .…”

Protein Function Microarrays Based on Self‐Immobilizing and Self‐Labeling Fusion Proteins

“…[10][11][12] A recent application of this strategy can be found in the expression of mutants of p53 as fusion proteins with a biotin acceptor sequence and their subsequent spotting and characterization on streptavidin-coated chips. [13] Another approach towards covalent immobilization of fusion proteins is based on the expression of proteins of interest as fusion proteins with the lipase cutinase. [14,15] Cutinase fusion proteins can be selectively immobilized on surfaces presenting a phosphonate-based suicide inhibitor and it has recently been shown that the approach can be used for the selective immobilization of cutinase antibody fusions.…”

Large-Scale Protein-Protein Interaction Detection Approaches: Past, Present and Future

“…Other limitations of utilizing Ni-His affi nity in functional microarrays arise from the fact that Ni-His interaction can be disrupted by washing, prolonged storage or commonly used reagents including EDTA and DTT (57). Slides coated with streptavidin bind biotinylated proteins very tightly and this system was used by Procognia to produce the fi rst commercially available functional microarray in 2002 (9). In addition, three-dimentional surfaces such as epoxy-modifi ed silicone elastomer microwells (108) can be used for microarrays.…”