PCR-Directed Protein In Situ Arrays
With the completion of the human genome sequence, the next priority is to identify the function of the thousands of proteins encoded within. One powerful technology that enables the high-throughput analysis of protein function is that of protein arrays. Protein arrays are usually produced by immobilizing many hundreds of individual proteins in a defined pattern onto a solid surface (1). Such arrays allow simultaneous screening of large numbers of proteins and permit parallel analysis of protein function. They can also be used to identify molecular interactions or, in the form of antibody arrays, to study protein expression profiling within patient samples (2). However, the main limitation to protein array technology currently is the production of the huge diversity of proteins that form the array elements. Many proteins, especially human proteins, are not expressed as functional molecules in heterologous hosts (3), and cloning of individual genes is also a time-consuming process. To overcome these problems, we have developed a cell-free protein array method, protein in situ arrays (PISA), which creates functional protein arrays directly from polymerase chain reaction (PCR) DNA by in vitro synthesis of individual tagged proteins on tag-binding surfaces, such that the tagged proteins are immobilized in situ as they are synthesized (4) (Fig. 1).
KeywordsMagnesium Urea Codon Agarose Electrophoresis
- 2.Michaud, G. A. and Snyder, M. (2002) Review: proteomic approaches for the global analysis of proteins. BioTechniques 33, 1308–1316.Google Scholar
- 6.Sawasaki, T., Ogasawara, T., Morishita, R., and Endo, Y. (2002) A cell free protein synthesis system for high-throughput proteomics. Proc. Natl. Acad. Sci. USA 99, 14, 652–14,657.Google Scholar
- 9.Taussig, M. J., Groves, M. A., Menges, M., Liu, H., and He, M. (2000) ARM complexes for in vitro display and evolution of antibody combining sites. In: (Shepherd, P. S., Shepherd, P. S., Dean, C. J., eds.) Monoclonal Antibodies: A Practical Approach. Oxford University Press, Oxford, UK: 91–109.Google Scholar