The PURE system for the cell-free synthesis of membrane proteins

Abstract

Cell-free gene expression systems are biotechnological tools for the in vitro production of proteins of interest. The addition of membrane vesicles (liposomes) enables the production of membrane proteins, including those in large-molecular-weight complexes, such as the SecYEG translocon or ATP synthase. Here we describe a protocol for the cell-free synthesis of membrane proteins using the protein synthesis using recombinant elements (PURE) system, and for subsequent quantification of products and analyses of membrane localization efficiency, product orientation in the membrane and complex formation in the membrane. In addition, measurements of ATP synthase activity are used as an example to demonstrate the functional nature of the cell-free synthesized proteins. This protocol allows the rapid production and the detailed analysis of membrane proteins, and the complete process from template DNA preparation to activity measurement can be accomplished within 1 d. In contrast to alternative methods using living cells, this protocol can also help to prevent the difficulties in membrane protein purification and the risks of protein aggregation during reconstitution into lipid membranes.

Relaxation dynamics of a compressible bilayer vesicle containing highly viscous fluid

“…Artificial cells with crowded internal environment are being developed in order to study the macromolecules in their physiological conditions [9,10]. Giant unilamellar vesicles (GUVs), which are cell-sized spherical shells consisting of a lipid bilayer, are commonly used as containers for artificial cells in which many synthetic chemical reactions take place.…”

Communicating artificial cells

“…One solution would be to incorporate membrane protein sensors and transporters within the artificial cells. Advances in cellfree expression have led to the reconstitution of the secYEG translocon, ATP synthase [59], multidrug transporter [60], pheromone sensor [61], and ion channels [62] in phospholipid vesicles. The surface of vesicles can also be decorated with antibodies in order to sense specific target cells.…”

Cell-free synthetic biology for in vitro prototype engineering

“…The choice of a well-characterised cell-free system almost entirely resides with E. coli platforms, which are based on either a crude cell extract [20–23] or a system of pu rified r ecombinant e lements (PURE) [24,25]. A vital area of importance to cell-free systems is the process of energy regeneration, which represents the major cost factor and limitation for both the PURE and cell extract-based routes.…”

“…This rather remarkable engineering feat is commercially available as the PURExpress® kit (New England Biolabs). While the high cost of the system prohibits scaled-up applications, a variety of cell-free researchers utilise the PURExpress® system to study the dynamics and kinetics of TX–TL [24,28–32]. The major advantage of the PURE system is it's high efficiency due to an absence of competing side reactions such as non-specific phosphatases [24], which rapidly degrade the energy source.…”