Functional Proteomics for Biomarker and Target Discovery
Joshua LaBaer, Harvard Medical School Institute of Proteomics
One of the most compelling steps in the post-genomic era will be learning the functional roles for all proteins. The Harvard Institute of Proteomics (HIP) has initiated a project to create a sequence-verified collections of full-length cDNAs representing human, human pathogen and model organism coding regions, in recombinational vector systems that allows the immediate in-frame transfer of all coding regions into virtually any protein expression vector. These transfers allow the addition of peptide tags to either or both end of the proteins. This repository, called the FLEXGene Repository (for Full-Length Expression-ready), is enabling the high-throughput (HT) screening of protein function for the entire set (or any customized subset) of genes using any method of in vitro or in vivo expression.
Using HT retroviral methods, these clones have been used to identify proteins capable of driving cell migration, altering the morphogenesis of normal epithelial structures, and affecting drug resistance in cells. A novel form of protein microarray, called nucleic acid programmable protein array (NAPPA), has been developed. This method substitutes the printing of proteins on the array with printing cDNAs encoding the proteins. Thus, the array is a DNA array that can be converted into a protein array by adding cell free protein synthesis machinery. This obviates the need to purify proteins, produces human proteins in a mammalian milieu, and avoids concerns about protein stability on the array because the proteins are made just-in-time for assay. NAPPA arrays can be used to study protein-protein interactions and protein-drug interactions, to identify vaccine candidates and to search for disease biomarkers in blood.
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