Cell-Free Synthesis of Macromolecular Complexes

Abstract

General rightsThis document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/pure/about/ebr-terms ABSTRACTCell-free protein synthesis based on E. coli cell extracts has been described for the first time more than fifty years ago. To date, cell-free synthesis is widely used for the preparation of toxic proteins, for studies of the translation process and its regulation as well as for the incorporation of artificial or labelled amino acids into a polypeptide chain. Many efforts have been directed towards establishing cell-free expression as a standard method for gene expression, with limited success. In this chapter we will describe the state-of-theart of cell-free expression, extract preparation methods and recent examples for successful applications of cell-free synthesis of macromolecular complexes.3 1/ Introduction -Motivation and challengesThe capacity of cell extracts to synthesize proteins has been shown in the fifties of the last century [1,2], several years before the identification of ribosomes as proteinsynthesizing machines [3]. The cell-free extract was based on the classical S30 fraction obtained by a 30,000 x g centrifugation step at 4°C for 1 hour. Initially, endogenous mRNA was used for in vitro translation [4]. Subsequently, Nirenberg and Matthaei developed a protocol to degrade endogenous messenger RNA present in the cell extract and to add exogenous mRNA [5,6]. The first cell-free protein synthesis (CFPS) from DNA, using a socalled coupled transcription-translation system was developed in the late sixties by the group of Zubay [7]. They used their coupled transcription-translation system to study the regulation of gene expression by the E. coli lactose operon. Most cell-free extract preparation and in vitro translation protocols are based on this protocol [8,9].Significant improvements with respect to protein yields were achieved in the late eighties, in particular by the group of Spirin, which established the use of phage-specific RNA polymerases, SP6 [10] or T7 RNA polymerases [8]. Using these polymerases a high level of a specific mRNA during the in vitro transcription-translation reaction can be achieved and maintained. Importantly, the Spirin laboratory described the first 'continuous' in vitro translation system. It allows for a continuous exchange of small molecules between a 'feeding compartment' providing energy and substrates (amino acids) for the translation reaction and a 'reaction compartment' from which inhibitory reaction products are removed by dialysis [10,11]. In a continuous set-up, the in vitro translation reaction can continue for several hours or even days, compared to 40-60 minutes using the classical reaction set-up.This allows obtaining significantly increased yields: for instance 6 mg chloramphenicol Cell-free expression has thus several very attractive applications, related to the expression of toxic proteins, rapid production of small q...