Membrane protein production in Escherichia coli cell‐free lysates

Abstract

a b s t r a c tCell-free protein production has become a core technology in the rapidly spreading field of synthetic biology. In particular the synthesis of membrane proteins, highly problematic proteins in conventional cellular production systems, is an ideal application for cell-free expression. A large variety of artificial as well as natural environments for the optimal co-translational folding and stabilization of membrane proteins can rationally be designed. The high success rate of cell-free membrane protein production allows to focus on individually selected targets and to modulate their functional and structural properties with appropriate supplements. The efficiency and robustness of lysates from Escherichia coli strains allow a wide diversity of applications and we summarize current strategies for the successful production of high quality membrane protein samples.

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

“…Third, the open environment of CFPS systems allows the incorporation of ncAAs with low solubility or poor cell-uptake [10]. Moreover, CFPS systems are not constrained by cell-viability, thus facilitating expression of toxic proteins or membrane proteins [11, 12]. Furthermore, the toxicity from overexpressing orthogonal translation components such as aminoacyl-tRNA synthetases, tRNAs, and ribosomes can be circumvented in CFPS systems [13].…”

In vitro synthesis of a Major Facilitator Transporter for specific active transport across Droplet Interface Bilayers

“…To study membrane protein function in vitro or to utilise them in synthetic biological systems it is generally necessary to extract the protein from this native membrane and reconstitute it into a detergent or lipid system, the properties of which can be more readily controlled. A variety of lipid structures have been used to replicate the membrane environment; from bicelles12 and nanodiscs34 to larger liposomes and giant unilamellar vesicles56. These vesicles provide a self-contained inner compartment that allows for the study of movement of molecules across the bilayer and are increasingly used as drug delivery systems in the medical field7.…”

The synaptic vesicle protein SV31 assembles into a dimer and transports Zn2+

“…In particular, integral membrane proteins are suitable targets (Henrich et al . ) as the complexity of expression is largely reduced, toxic effects are minimized and restrictions in targeting and translocation are eliminated. The open accessibility of CF reactions and their high tolerance for numerous additives (Hein et al .…”

“…In the field of synthetic biology CF expression systems emerge as a potent tool for the production of proteins difficult to express. In particular, integral membrane proteins are suitable targets (Henrich et al 2015b) as the complexity of expression is largely reduced, toxic effects are minimized and restrictions in targeting and translocation are eliminated. The open accessibility of CF reactions and their high tolerance for numerous additives (Hein et al 2014) allows completely new approaches to synthesize membrane proteins in the presence of detergents [detergent-based cell-free expression (D-CF) mode] or supplied artificial bilayers [lipid-based cell-free expression (L-CF) mode].…”