Co-translational association of cell-free expressed membrane proteins with supplied lipid bilayers

Abstract

Routine strategies for the cell-free production of membrane proteins in the presence of detergent micelles and for their efficient co-translational solubilization have been developed. Alternatively, the expression in the presence of rationally designed lipid bilayers becomes interesting in particular for biochemical studies. The synthesized membrane proteins would be directed into a more native-like environment and cell-free expression of transporters, channels or other membrane proteins in the presence of supplied artificial membranes could allow their subsequent functional analysis without any exposure to detergents. In addition, lipid-dependent effects on activity and stability of membrane proteins could systematically be studied. However, in contrast to the generally efficient detergent solubilization, the successful stabilization of membrane proteins with artificial membranes appears to be more difficult. A number of strategies have therefore been explored in order to optimize the co-translational association of membrane proteins with different forms of supplied lipid bilayers including liposomes, bicelles, microsomes or nanodiscs. In this review, we have compiled the current state-of-the-art of this technology and we summarize parameters which have been indicated as important for the co-translational association of cell-free synthesized membrane proteins with supplied membranes.

Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination

“…It is, of course, necessary in the cell-free system of the precipitate type to solubilise the precipitated protein and to refold it as a prelude to purification and crystallisation. The option is always there however, to perform expression in the presence of detergent micelles [34], liposomes [35] or nanodiscs [36], which act as membrane mimetic receptacles for the nascent protein, with a view to diverting it, partly at least, away from aggregation. These alternative approaches however, usually require additional steps where the ‘reconstituted’ protein is isolated from aggregated material and the protein is removed or exchanged from its membrane mimetic and reformulated in preparation for crystallisation screening.…”

“…However, it is possible to do the expression in the presence of a membrane mimetic for the direct production of functional protein. To date, detergent micelles [46], liposomes [35], nanodiscs [47] and bicelles [48] have been used for this purpose, and each has its own pros and cons. As a logical extension to the current work, we are proposing to use the bicontinuous lipidic mesophase as an alternative receiving membrane mimetic, for several reasons.…”

Nanodiscs in Membrane Biochemistry and Biophysics

“…With certain proteins and detergents, some atypical difficulties may be faced due to their specific properties. For instance, the binding of some hydrophobic components to BioBeads® was reported 32,33 when cell-free expressed membrane protein was incorporated in Nanodiscs, and an alternative sorbent was used to improve the overall yield of incorporated target. The extent of such side reactions may also strongly depend on the relative amount of all components present, the conditions of assembly and the time span used for detergent removal.…”

Single-channel electrophysiology of cell-free expressed ion channels by direct incorporation in lipid bilayers

“…Bacterial lysates that contain the ribosomal machinery and are supplemented with amino acids, a metabolic energy supply and a protein-encoding plasmid, have been shown to express a large variety of membrane proteins, either as precipitates or solubilized in detergent micelles. [12][13][14] Interestingly, when liposomes are added to the cell-free reaction mixture, spontaneous reconstitution has been demonstrated for a variety of cell-free expressed membrane proteins, including stearyl-CoA desaturase, glucan synthase, ATP synthase, DesK thermosensor, endothelin receptors A and B, bacteriorhodopsin, connexin-43, aquaporin Z, and the ion channels Kcv and KcsA. [15][16][17][18][19][20][21][22][23][24] Given that incorporation of protein into the liposome cannot be facilitated by translocon components as these are not present in the lysate, it has been postulated that the presence of detergents, trace amounts of native lipids, or a close ribosome-liposome proximity aids protein insertion in the lipid bilayer of the liposomes.…”

“…[15][16][17][18][19][20][21][22][23][24] Given that incorporation of protein into the liposome cannot be facilitated by translocon components as these are not present in the lysate, it has been postulated that the presence of detergents, trace amounts of native lipids, or a close ribosome-liposome proximity aids protein insertion in the lipid bilayer of the liposomes. [12][13][14] In principle, the bilayer self-insertion of cell-free expressed membrane proteins can be exploited as a purication method for ion channel electrophysiology, as demonstrated in a small number of recent studies. [25][26][27][28][29] For example, large unilamellar vesicles containing the self-inserted ion channels VDAC or MscL have been puried from the expression mixture by density gradient centrifugation and subsequently fused with giant unilamellar vesicles (GUVs).…”