# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 5 Cell-Free Expression for Reinsertion of Membrane Proteins before or after Formation of tBLMs  
One of the major difficulties with regard to the reconstitution of MPs in tBLMs is the possibility of preparing proteoliposomes which retain their functional activity for their use during the vesicle fusion step. In addition, the material losses and/or the possible protein denaturation that could be encountered during this latter step, when using classical \"leaflet-by-leaflet\" formation of tBLMs in the \"bottom-up\" approach, leads more often to a very low amount of proteins which is finally reinserted into the tBLMs, and at the end of the day, does not allow to measure any protein activity. Figure 5: Principle of formation of _protein-tBLMs_ or ptBLMs. A recombinant membrane protein with a ‘_His-tag_’ in C- or N-terminus solubilized in detergent micelles is first attached by affinity to nitrilotriacetic acid (NTA) moiety grafted to a metal surface in the presence of Ni\({}^{2+}\) (or possibly Cu\({}^{2+}\)) cations. The lipid bilayer surrounding the attached protein is obtained in a second step by in situ dialysis or using microporous biobeads in the presence of lipid–detergent mixed micelles (adapted with permission from [107]. Copyright 2004, American Chemical Society, and with permission from [132]. Copyright 2008, American Vacuum Society). To address this issue, the reconstitution of integral MPs produced in vitro by cell-free expression synthesis directly into preformed tBLMs has been proposed, whatever the tethering molecules used e.g., polyethylene glycol (PEG) [39], _telechelics_[38], peptides [37; 20; 25; 230; 252] or proteins [253]. The basic idea is to bypass the difficult expression, purification and reconstitution procedures inherent to dealing with integral MPs. The production of recombinant membrane proteins by classic overexpression techniques presents some limiting features (i.e., low expression rate and/or cytotoxicity for the host cells). Another common problem is the detergent-based purification and refolding of MPs, possibly aggregated, in phospholipid environment like liposomal membranes into their native functional conformation. Cell-free transcription/translation systems are based on gene expression in the presence of cell extracts of various species [254; 255; 256; 257; 258; 259; 260; 261] like bacteria (e.g., _E. coli_), insects (e.g., _Spodoptera frugiperda_), plants (e.g., _wheat germ_) or eukaryotes (e.g., rabbit reticulocytes). The relevant gene, provided in the form of cDNA (complementary deoxyribonucleic acid), is mixed with the cell lysate and the translated protein directly inserts in the membrane systems provided, generally liposomes, nanodisks or amphipols (a class of amphiphilic polymers designed to keep membrane proteins soluble in water without the need for detergents). One of the advantages of these in vitro systems over classic in vivo systems, is their ability to synthesize cytotoxic MPs, regulator or unstable proteins. Moreover, they are completely open systems, allowing each parameter of the reaction (such as pH, redox potential, ionic strength etc.) to be modified depending on the target protein. They offer the possibility of the integral proteins to spontaneously reincorporate in lipid membranes in a functional orientation and sufficient amount. Currently, many research workers have opted for these synthesis systems as tools for structural and functional studies [261; 262]. Therefore, a number of membrane proteins have been successfully reincorporated in tBLMs.