Proteomics and biotechnology studies require simple and rapid methods to convert the genetic information into proteins. Whereas heterologous protein expression in living cells is a time-consuming process, in vitro translation directs protein synthesis in hours from added linear PCR DNA without the need for E. colt cloning, thus providing an attractive alternative to cell-based methods for high throughput production of proteins. Moreover, the open nature of cell-free systems and availability of various prokaryotic and eukaryotic cellular lysates offers a flexible choice of conditions for synthesis of folded proteins or production of "difficult" proteins that are not possible by in vivo systems. Finally, cell-free extracts express protein populations in a single reaction, allowing for the development of powerful proteomic tools. This article will review the recent advances in cell-free protein expression and its applications in biotechnology, proteomics and fundamental biological research. (C) 2010 Elsevier Ltd. All rights reserved.