Protein expression profiling using protein microarray

Abstract

Protein microarray has so many advantages for the high-throughput analysis of protein. In contrast to conventional protein analysis methods, only minute amounts of sample is required, little bias toward protein’s property is exist and multiplex analysis is possible in the analysis with protein microarray. In this work, three kinds of protein microarrays were developed. Firstly antibody microarray composed of 30 antibodies was constructed. These antibodies were developed in rabbit against proteins related with amino acid biosynthetic pathway of Escherichia coli. Several critical factors affected to performance of the antibody microarray were investigated and of them, cross-reactivity of antibody was found to manly cause the deviations of result from antibody microarray. With correction factors to offset the deviations and two color fluorescence assay, protein expression level of L-threonine-overproducing mutant (TF5015) was compared with that of wild-type (W3110). In TF5015, ten proteins were identified as up-regulated proteins and one was as down-regulated protein. This profiling result was validated with conventional analysis tool, western blotting and two-dimensional gel electrophoresis. Next allergen microarray was developed to diagnose the allergy. The major symptom of allergy is the elevation of allergen-specific IgE concentration in serum. Thus this allergen microarray was used to detect IgE expression level in patients’ sera for diagnosing. Extracts of two mites were used as model allergens and spotted onto glass slide. Subsequent binding of biotinylated anti-human IgE and streptavidin-Cy3 was employed as a fluorescence-producing method. To get precise and reproducible results, some analysis factors were optimized, and under optimized conditions, 43 human sera were profiled. Profiling results were compared with conventional allergy diagnosis method, CAP system assay, and showed improved reproducibility and reliability compared with previous reported resu...