Functional Cell-free Synthesis of a Seven Helix Membrane Protein: In situ Insertion of Bacteriorhodopsin into Liposomes

Kalmbach, Rolf; Chizhov, Igor; Schumacher, Miria C.; Friedrich, Thomas; Bamberg, Ernst; Engelhard, Martin

doi:10.1016/j.jmb.2007.05.087

Cited by 139 publications

(117 citation statements)

References 51 publications

(48 reference statements)

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“…We confirmed that the cell-free product in the presence of SUV liposomes had the proper fold. The total amount of total BR was about 80-fold larger than that of the previous study (0.04 mg/mL RM 16 ). On the other hand, the ratio of folded BR, which was estimated by visible absorption for the folded BR and SDS-PAGE band intensity for the total BR (see Materials and Methods), was lower [$15% (Fig.…”

Section: Screening Of Detergents For Functional Cell-free Expression contrasting

confidence: 75%

“…16 Therefore, we examined the synthesis of this construct in the dialysis mode, in the presence of egg PC SUV liposomes. The products (3.2 mg/mL RM) were obtained as a reddish-purple precipitate.…”

Section: Screening Of Detergents For Functional Cell-free Expression mentioning

confidence: 99%

“…[15][16][17][18][19][20][21][22] The liposome, a synthetic lipid bilayer, provides a more natural environment than the detergent micelle. The most suitable lipids can be selected for individual proteins.…”

Section: Introductionmentioning

confidence: 99%

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Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

et al. 2009

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Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane a-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.

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Section: Screening Of Detergents For Functional Cell-free Expression contrasting

confidence: 75%

Section: Screening Of Detergents For Functional Cell-free Expression mentioning

confidence: 99%

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Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

et al. 2009

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“…[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. [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.…”

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confidence: 99%

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

Friddin

Smithers

Beaugrand

et al. 2013

Analyst

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Single-channel electrophysiology with lipid bilayer systems requires ion channel expression, purification from cell culture, and reconstitution in proteoliposomes for delivery to a planar bilayer. Here we demonstrate that single-channel current measurements of the potassium channels KcsA and hERG S5-S6 can be obtained by direct insertion in interdroplet lipid bilayers from microliters of a cell-free expression medium.Electrophysiology is the gold standard for the functional characterization of ion channel proteins, including screening of new drugs that target this pharmaceutically important class of membrane proteins. 1,2 The patch clamp technique involves intimate contact of an electrode-containing glass pipette with the membrane of a cell in which the channel of interest is overexpressed, and is used most widely for ensemble current measurements of channel populations. The alternative 'bilayer lipid membrane' (BLM) approach, in which puried ion channels are introduced by proteoliposome fusion into a pure-lipid membrane that separates two electrode-containing aqueous compartments, is typically employed for current measurements of single channels. 3,4 Automated instruments for mediumthroughput patch clamp electrophysiology have entered the market in the last decade, 5,6 whereas systems for automated and/or parallel BLM electrophysiology are under development by several research groups and companies. [7][8][9] Although there are recent developments toward patch clamping of giant proteoliposomes, 10 patch clamping typically relies on eukaryotic cells that are able to overexpress the ion channel of interest with concomitant cell membrane incorporation. 2 However, this is not possible when the expressed channel aggregates or when membrane incorporation is toxic to the cell. Proteoliposome formation, for patch clamping or for the BLM method, also requires ion channel overexpression, but, at the cell culture stage, not necessarily as solubilized or membrane-incorporated protein, and is not restricted to eukaryotic cells. 3 This offers more options to obtain the desired ion channel but at the cost of having to purify it from cell culture, with subsequent reconstitution from detergent solution into liposomes, which is a laborious process that requires a relatively high protein yield.In recent years, considerable progress has been made with the cell-free expression of membrane proteins, 11 which bypasses any cytotoxicity problems and facilitates protein purication. 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. 12-14 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 rece...

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“…This was accomplished by simultaneous cell-free expression of an apolipoprotein and BO in the presence of lipids and all-trans retinals, leading to the self-assembly of BR-containing nanolipoprotein particles (NLPs) [54]. In addition, high-yield cell-free synthesis of BR in the presence of small unilamellar liposomes has been demonstrated [122].…”

Section: Site-directed Isotope Labeling and Cell-free Expression Of Brmentioning

confidence: 99%

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

Rothschild

2016

BSI

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Abstract. Membrane proteins facilitate some of the most important cellular processes including energy conversion, ion transport and signal transduction. While conventional infrared absorption provides information about membrane protein secondary structure, a major challenge is to develop a dynamic picture of the functioning of membrane proteins at the molecular level. The introduction of FTIR difference spectroscopy around 1980 to study structural changes in membrane proteins along with a number of associated techniques including protein isotope labeling, site-directed mutagenesis, polarization dichroism, attenuated total reflection and time-resolved spectroscopy have led to significant progress towards this goal. It is now possible to routinely detect conformational changes of individual amino acid residues, backbone peptides, binding ligands, chromophores and even internal water molecules under physiological conditions with time-resolution down to nanoseconds. The advent of ultrafast pulsed-IR lasers has pushed this time-resolution down to femtoseconds. The early development of FTIR difference spectroscopy as applied to membrane proteins with special focus on bacteriorhodopsin is reviewed from a personal perspective.

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Functional Cell-free Synthesis of a Seven Helix Membrane Protein: In situ Insertion of Bacteriorhodopsin into Liposomes

Cited by 139 publications

References 51 publications

Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

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

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

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