In the past decade, biological research has witnessed a paradigm shift from focused reductionist approaches to a greater dependence on large """"""""industrial-sized"""""""" projects. High-throughput (HT) biology began in earnest with the Human Genome Project, and increasingly these HT tools and approaches are being exploited for protein research. Given the importance of proteins in disease etiology and treatment, a major challenge facing biology is the elucidation of the physiological role of all proteins. In this light, the field of functional proteomics, a new approach to the HT study of proteins, will enable the expression and subsequent assay of proteins and their various properties such as subcellular location, interacting partners, biochemical activity or regulated modification at a scale of thousands at a time. A prerequisite for this approach is the need for large collections of cDNAs in a format conducive to HT protein expression. We and others (Walhout 2000, Brizuela 2001) have begun to create such collections of cDNAs using the novel technology of recombinational cloning that allows rapid transfer of DNA fragments from one vector to another, in frame and without mutation. However, high-quality collections of human clones are not yet available. Here we propose to build a collection of 1000 expression-ready human cDNA clones representing genes of significance to breast cancer (BC 1000). We are nearing completion of 100 such clones assembled in a pilot study and have already found them to be invaluable in the development of HT methods for both in vitro and in vivo studies. However, the relatively modest size of the collection prevents its application in any meaningful screening experiments. Thus it is important to expand this collection to a size that will enable more comprehensive screens and the development of experimental technologies that will truly exploit the HT setting. In order to exploit this resource, we have developed a novel method for creating protein microarrays that enables the HT functional study of proteins. This approach, called Nucleic Acid-Programmable Protein Array (NAPPA), replaces the complex process of spotting purified proteins with the simple process of spotting DNA. By exploiting the recombinational format of the BC1000, genes are then simultaneously transcribed/translated in a cell-free system and the resulting proteins are immobilized in situ, minimizing direct manipulation of the proteins and making this approach well suited to HT applications. Advantages of this approach include: the ability to express and interact proteins in a mammalian milieu, no requirement for HT expression-purification-storage of proteins, and real time collection of data, minimizing concerns about protein stability. We propose to adapt this method to a glass matrix and miniaturize it to allow for the screening of thousands of proteins simultaneously. We will demonstrate the effectiveness of this approach by executing a 1000 x 1000 interaction matrix with the BC1000.
| LaBaer, Joshua; Ramachandran, Niroshan (2005) Protein microarrays as tools for functional proteomics. Curr Opin Chem Biol 9:14-9 |
