production of membrane proteins using cell-free expression systems

REVIEW

Production of membrane proteins using cell-free

expression systems

Daniel Schwarz, Volker Dötsch and Frank Bernhard

Centre for Biomolecular Magnetic Resonance, University of Frankfurt/Main,Institute for Biophysical Chemistry, Frankfurt/Main, Germany

Production of membrane proteins (MPs) is a challenging task as their hydrophobic nature andtheir specific requirements in cellular expression systems frequently prevent an efficient syn-thesis. Cell-free (CF) expression systems have been developed in recent times as promising toolsby offering completely new approaches to synthesize MPs directly into artificial hydrophobicenvironments. A considerable variety of CF produced MPs has been characterized by functionaland structural approaches and the high success rates and the rapidly accumulating data onquality and expression efficiencies increasingly attract attention. In addition, CF expression is ahighly dynamic and versatile technique and new modifications for improved performance as wellas for extended applications for the labeling, throughput expression and proteomic analysis ofMPs are rapidly emerging.

Received: February 21, 2008Revised: April 15, 2008Accepted: May 1, 2008

Keywords:

Cell-free protein synthesis / High-throughput proteomics / Protein expression analysis /Stable isotope labelling / Transmembrane proteins

Proteomics 2008, 8, 3933–3946 3933

1 Introduction

A variety of intrinsic characteristics make membrane pro-teins (MPs) to be one of the most problematic targets inexpression studies. The hydrophobic nature of MPs oftenpromotes unfolding and aggregation that is frequently fol-lowed by proteolysis. Targeting, translocation, and stableintegration of MPs into native cellular membranes are keyproblems and often represent the bottlenecks in expressionapproaches [1, 2]. Moreover, disintegration and destabiliza-

tion of host cell membranes as well as toxic effects due tosystem overloads or to transport and pore forming activitiesof the recombinant MPs could cause further tremendousproblems. As a result, the functional and structural charac-terization of MPs still lacks far behind to that of cytoplasmicproteins.

Cell-free (CF) expression systems have emerged in recenttimes as promising tools in order to accelerate and tostreamline MP expression approaches [3–6]. The eliminationof a living host environment during protein overexpressionin combination with the open accessibility of the reactionoffers a variety of valuable advantages. It is evident thatproblems with toxic or inhibitory effects of the recombinantMPs to the host cell physiology are minimized or even com-pletely eliminated. The expression reaction is not enclosedby membranes and any compound can thus be directlyadded without considering transport or metabolic conversionproblems. Protease inhibitors, ligands, cofactors, or sub-strates can be considered as possible additives that might behelpful in stabilizing the freshly translated MPs. Compli-cated transportation or translocation systems for the synthe-sized MPs are no longer required as most native membraneshave been removed from the CF extracts during the prepa-

Correspondence: Dr. Frank Bernhard, Centre for BiomolecularMagnetic Resonance, University of Frankfurt/Main, Institute forBiophysical Chemistry, Max-von-Laue-Str. 9, D-60438 Frankfurt/Main, GermanyE-mail: [email protected]: 149-69-798-29632

Abbreviations: CECF, continuous exchange cell-free; CF, cell-free;CMC, critical micellar concentration; DDM, n-dodecyl-b-D-malto-side; FM, feeding mixture; GPCR, G-protein coupled receptor;MP, membrane protein; NOE, nuclear overhauser effect; RM,

reaction mixture; RRL, rabbit reticulocyte lysates; TMS, trans-membrane segment; WG, wheat germ system

DOI 10.1002/pmic.200800171

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

3934 D. Schwarz et al. Proteomics 2008, 8, 3933–3946

ration procedure. In contrast, the MPs can be maintainedsoluble in artificial hydrophobic environments like detergentmicelles [3, 7, 8]. Furthermore, CF expression reactions arecarried out in small volumes of few milliliters or evenmicroliters and incubation times of few hours are alreadysufficient for the production of milligram amounts of MPs.Altogether, these properties make CF expression systemsinteresting for the preparative scale production of MPs aswell as for throughput screening and proteomics ap-proaches.

2 CF expression systems

Although known already for several decades for the analyti-cal scale expression of proteins, the CF preparative scaleproduction became possible in rather recent times afterdeveloping new reaction designs and protocols [9, 10]. CFexpression systems have already become routine techniquesfor the production of soluble proteins for structural analysis[11–14]. However, the preparative scale CF production ofMPs is still a very new application that has been developedduring the last 5 years. Despite this relatively short period,the recently published first X-ray structure of an MP by usingalso CF produced samples indicates a promising perspective[15]. CF expression already represents a diverging pool oftechniques. Different extract sources have been establishedand several system configurations and expression modeshave been developed that significantly can impact the qualityand performance of individual MP expressions.

2.1 Extract sources

Still only few sources are currently considered for CF extractpreparations. Most common are extracts from Escherichia coliand from wheat germ embryos. The popular E. coli S30extract is obtained by centrifugation of lysed cells at30 0006g and it includes most enzymes necessary for pro-tein synthesis [16, 17]. Endogenous mRNA as well as all lowmolecular weight compounds are eliminated from theextract during the preparation procedure. This helps to sup-press background expression and the operator has further-more complete control over the amino acid pool in the reac-tion, which is of importance for the efficient labeling ofexpressed MPs. Reactions with E. coli S30 extracts are routi-nely set up as coupled transcription/translation systems anddsDNA has to be added as a template (Fig. 1). Not only plas-mid DNA but also linear DNA from PCR reactions can beused, and this option could considerably accelerate through-put applications. E. coli CF expression systems could eitherbe established according to published protocols [3, 18], oralternatively commercial kits from companies like RocheApplied Science, Invitrogen, or Qiagen might be considered.

E. coli CF expression systems are very efficient for theproduction of even large eukaryotic MPs, but they neitherprovide most PTMs nor specific eukaryotic folding systems.

Figure 1. Versatility of CF expression systems for the productionof MPs. The most commonly used reaction types (translation andcoupled transcription/translation), reaction configurations (batchand CECF), and expression modes (P-CF, D-CF, L-CF) are illustrat-ed.

CF expression systems based on wheat germ extracts couldtherefore offer an alternative option in order to address thosepotential problems. Wheat germ systems (WGs) are fre-quently operated as translation systems with suppliedmRNA as template as the Mg21 optima for transcription andtranslation are different (Fig. 1). The high stability of theextract compounds allows the extension of reaction times foreven weeks with final protein yields of upto 10 mg/mL atoptimal conditions [12]. Wheat germ extracts are relativelydifficult to prepare as the embryo is surrounded by theendosperm containing high levels of protein and nucleicacids degrading enzymes as well as translation inhibitors. Inaddition, high batch variations in the extract quality of indi-vidually prepared extracts exist that primarily depend on theorigin and source of the wheat germs. However, commercialsuppliers of wheat germ extracts or expression systems areavailable such as Cell-Free Sciences or Roche Applied Sci-ence.

Further extract sources or alternative CF expression sys-tems exist but only few have been used so far for the pro-duction of MPs. The PURE (protein synthesis using recom-binant elements) system represents a nearly completereconstituted translation machinery of all aminoacyl-tRNA-synthetases and translation factors that have been separatelyproduced and purified from conventional in vivo E. coli


Proteomics 2008, 8, 3933–3946 Technology 3935

expression [19, 20]. The ribosomes are still isolated from E.coli extract, but with a significantly higher purity after cen-trifugation at 100 0006g. The PURE system allows CF syn-thesis under better defined conditions which can be advan-tageous for the study of folding pathways or translationkinetics. However, expression yields are lower if comparedwith other CF expression systems and it is only used for theanalytical scale production of proteins. Further options areCF expression systems based on rabbit reticulocyte lysates(RRLs) [21] or on insect cell extract [22]. The codon usage ofRRL systems is strongly biased and optimized for globinsynthesis. Relatively high endogenous levels of ribonucleasemight cause further problems. PTMs and in particular gly-cosylation of MPs might be relatively efficient in these sys-tems, but both are only used for the analytical scale MP pro-duction.

2.2 Template design and reaction compounds

In coupled transcription/translation systems, template DNAhas to be provided under control of a specific and strongpromoter sequence, most commonly recognized by phagepolymerases like T7, SP6, or T3 RNA-polymerase. For E. coliCF expression systems, the target genes are placed undercontrol of the T7 promoter. Some suitable vectors are pET(Novagen) or pIVEX (Roche Diagnostics) derivatives. Theproductivity of prokaryotic systems strongly depends on therate of the initiation of translation. N-terminal tags like theT7-tag or poly(His)6-tag or smaller fusion proteins like thio-redoxin could therefore considerably enhance the expressionin particular of eukaryotic MPs [23]. This sometimes has aquite dramatic positive effect of terminal peptide tags andhas previously also been reported for the CF expression ofsoluble proteins [24]. The presence of complex secondarystructures in the 50-untranslated regions of mRNAs mayfurther inhibit translation by preventing contacts to theribosomes, e.g., by masking the ribosome binding site.Removing potentially unfavorable sequences after scanningof the mRNA sequence with corresponding algorithms likethe commercial ProteoExpert program could significantlyincrease and optimize expression results [25, 26]. Stable viralleader sequences are usually employed in WGs in order tosubstitute for the otherwise required capping and poly(A)structures. A decrease in translation as a result of decappedmRNAs and of degraded poly(A) tails during prolongedincubation is then avoided [12].

Besides the extracts, CF expression reactions contain avariety of essential compounds with sometimes well definedand critical concentration optima (Table 1). Divalent Mg21

ions are essential for many biological reactions and their freeconcentration in CF extracts is a very hard to determinevalue. For maximum protein production yields, it is thereforeadvisable to optimize Mg21 concentration for each new batchof prepared extract or even freshly prepared stock solution.For E. coli systems, the total Mg21 ion concentrations typi-cally range from 8 to 20 mM, while eukaryotic translation

Table 1. Reaction components of commonly used CF systems

Component PANOx-SP Cytomim E. coli Wheat germ

Reaction type Batch Batch CECF CECFSource of extract E. coli E. coli E. coli Wheat

embryosTemplate DNA DNA DNA mRNA

Basic components

NTPs/NMPsa) A/C/G/U A/C/G/U A/C/G/U A/GSource of tRNA E. coli E. coli E. coli YeastTwenty Amino acids 1 1 1 1

Mg21 acetate 1 1 1 1

Mg21 glutamate 1 1 2 2

K1 acetate 1 1 1 1

K1 glutamate 1 1 2 2

Folinic acid 1 1 1 2

Tris pH 8.2 1 1 1 1

HEPES pH 8.0 2 2 1 1

DTT 1 1 1 1

Enzymes

Pyruvate kinase 1 1 1 2

Creatine kinase 2 2 2 1

T7-RNA polymerase 1 1 1 2

RNAse inhibitor 2 2 1 1

Disulfide isomerase 1 1 2 2

Energy system

Phosphoenol pyruvate 1 2 1 2

Sodium pyruvate 2 1 2 2

Acetyl phosphate 2 2 1 2

Creatine phosphate 2 2 2 1

NAD 1 1 2 2

CoA 1 1 2 2

Na1 oxalate 1 1 2 2

Additives

Putrescine 1 1 2 1

Spermidine 1 1 2 1

PEG 8.000 2 2 1 2

Glycerol 2 2 2 1

DTT 2 2 1 1

NaN3 2 2 1 1

Protease inhibitor mix 2 2 1 1

a) Optional phosphate donor in PANOx/Cytomin systems usingalternative energy regeneration sources.

systems include only 2–4 mM Mg21 [27]. Potassium andammonium acetate or the corresponding glutamates can beadded into E. coli systems at high concentrations exceeding200 mM. WGs often contain 100 mM potassium acetate [12,27]. ATP and GTP are sufficient to provide the requiredenergy for mRNA translation in WGs, while the fournucleoside triphosphates ATP/CTP/GTP/UTP are necessaryin the coupled transcription/translation reaction type of E.coli CF expression systems. Amino acids are supplied inconcentrations between 0.3 and 2 mM while even higheramounts of unstable amino acids (e.g., R, C, W, M, D, E)



could significantly increase expression. MPs often have astrong bias in amino acid composition and adjusting theamino acid pool of the reaction accordingly could furtherresult in higher expression yields. Problems with the codonusage upon expression of heterologous genes could beaddressed by modulating the total tRNA concentration or byaddition of rare codon-enriched tRNA. An alternative optionwould be to use synthetic genes designed according to thepreferred codon usage of the selected CF expression system.

NTPs are essential energy sources and they are rapidlyconsumed in CF expression reactions. In addition, bacterialcell extracts have high endogenous phosphatase and ATPaseactivities resulting in further uncoupled NTP hydrolysis.Energy supply is therefore the main limiting factor for CFsynthesis and a number of modified protocols for E. coli sys-tems have been developed that as a major variation use dif-ferent energy sources and pathways for the efficient ATPregeneration (Table 1). In conventional protocols, high-energy phosphate donors like phosphoenol pyruvate, acetylphosphate, or creatine phosphate are supplied as secondaryenergy substrate together with the corresponding kinases[10, 28]. Excess of phosphate generated by the breakdown ofenergy sources and nucleotides has been identified as an in-hibitor of translation. In particular in batch configurations,the strategy of energy regeneration is of importance as accu-mulation of inhibitory phosphate should be minimized.Proposed modified energy regeneration pathways for batchsystems use the oxidation of substrates from the glycolyticpathway (e.g., pyruvate, glucose-6-phosphate, glucose) for thegeneration of ATP concomitant with the consumption ofreaction by-products [29]. The “PANOx” system combinesphosphoenol pyruvate as the conventional energy sourcewith the addition of nicotinamide adenine dinucleotide andcoenzyme A in order to use in addition to pyruvate for ATPregeneration [30]. Protocols using alternative nonpho-sphorylated energy sources like pyruvate in the “Cytomim”system have further been developed (Table 1) [31]. Oxidativephosphorylation, the most efficient natural source of ATP, isused and potassium and magnesium glutamate as well asthe polycations spermidine and putrescine should also moreclosely mimic a cytoplasmic composition of the reaction.Having efficient energy regenerating systems, the relativelyexpensive NTPs could also be replaced by NMPs in order togenerate a more cost-effective system [29].

A powerful option for CF synthesis is to include certainadditives that might stabilize the synthesized MP or facilitateits functional folding. The CF expression reaction is easilyaccessible at any time point, so even refeeding of criticalsubstances could be considered [32]. PEGs can be supple-mented to induce a molecular crowding effect for betterreaction conditions. Reducing conditions can be modulatedby addition of substances like DTT which in addition keepsthe T7 RNA polymerase in its reduced and active form. Onthe other hand, rather oxidative conditions can be chosen inorder to support the formation of disulfide bridges of thesynthesized proteins.

2.3 Configuration of CF reactions

The least complicated configuration for CF expression is aone compartment batch reaction (Fig. 1). Depending on theprotocol, the reaction times are limited to few hours or evenless due to rapid shortage of precursors and accumulationof inhibitory by-products. The consequences are relativelylow yields, while optimized protocols and reaction set-upscan be efficient enough to produce close to 1 mg of recom-binant protein per 1 mL of reaction [33]. With appropriatevolumes even sufficient amounts of MPs for structuralapproaches can be obtained as recently demonstrated forthe crystallization of the CF produced seleno-methioninelabeled multidrug transporter EmrE [15]. Batch reactionsare ideal for throughput expression screens in small scaleswith reaction volumes as low as only few microliters. Theset-up and further processing of the reaction can be carriedout in microplate formats and the whole process could beautomated with implementation of modified pipettingroboters.

A remarkable strategy to optimize the efficiency of CFreactions was the design of continuous exchange CF (CECF)systems [9]. The principle is to separate the reaction into twocompartments that are separated by a semipermeable mem-brane (Fig. 1). One compartment holds the reaction mixture(RM) with all high molecular weight compounds like the CFextract, enzymes, and nucleic acids. The second compart-ment provides a reservoir of low molecular weight pre-cursors like NTPs, energy sources, and amino acids andrepresents the feeding mixture (FM). The molecular weightcut-off of the separating membrane is usually relatively highand in between 10 and 50 kDa. It has to be emphasized thateven at higher pore sizes no significant leakage of essentialproteins from the translation machinery of the extract occursfrom the RM into the FM as they form large complexesthrough multiple interactions. Synthesized MPs also stay inthe RM as the insertion into detergent micelles usuallyincreases the molecular weight of even small proteins above50 kDa. Efficient substance exchange between the two com-partments through the membrane is ensured by vigorousstirring or shaking during incubation. The reaction time issignificantly extended by the continuous supply of freshprecursors from the FM into the RM concomitant with thecontinuous dilution of inhibitory by-products from the RMinto the FM. The volume ratio of RM/FM is usually in be-tween 1:10 and 1:50 and it is an important parameter for thefinal yield of recombinant protein. Higher FM volumes willcertainly increase product yields, but not in a linear correla-tion. The exchange rate of compounds between the twocompartments as well as the capacity of the CF extract willbecome more and more limiting and reduce the gain of yield.Considering the relatively high costs of the precursors in theFM, a ratio of approximately 1:20 may be a reasonable com-promise for maintaining CF expression for 6–8 h with finalyields of up to several milligrams recombinant protein per1 mL of RM.



Since CECF reactions have to be equipped with a mem-brane, their setup and handling is relatively complicated andlimits their use in throughput applications. A bilayer systemhas recently been proposed as an interesting alternative con-figuration in particular for throughput purposes [34]. Thebilayer system resembles a simplified version of the CECFsystem, in which the RM is carefully overlaid by the FMwithout separation by a membrane. Substances between thetwo layers exchange simply by diffusion and translation canbe prolonged for several hours yielding submilligramamounts of protein which is sufficient for functional analy-sis. The reaction can be set up in microplates and a ratio ofRM/FM of approximately 1:5 is recommended for best per-formance.

3 CF reaction modes for MP production

3.1 CF production of MPs as precipitates (P-CF)

Besides CF expression systems (e.g., E. coli vs. wheat germ)and configurations (e.g., batch vs. CECF), the choice of thereaction mode is an important consideration in particular forthe production of MPs. A standard CF expression reaction isdevoid of substantial amounts of membranes or lipids. Mostmembrane fragments originating from the lysed cells havebeen removed by centrifugation steps. However, lipids arenot completely depleted and residual amounts of approxi-mately 50–100 mg/mL of extract can be detected. Those lipidsmight be associated with other proteins and it is not clearwhether they are organized in membranes or vesicles. As aresult, no substantial amounts of hydrophobic environmentsare present to keep MPs soluble after translation. Thisstandard CF expression mode for MP production will there-fore result in MP precipitates (P-CF, Fig. 1). The P-CF modereminds the formation of inclusion bodies upon MP expres-sion in E. coli cells. However, there are some important dif-ferences: (i) precipitates are always formed during the suc-cessful P-CF expression of MPs, while inclusion body for-mation is a much rarer event mostly observed with outerMPs. (ii) P-CF generated precipitates can efficiently be solu-bilized with detergents by gentle shaking for few hours [5]. Incontrast, inclusion bodies usually have to be solubilized withstrong denaturants like high concentrations of urea or gua-nidinium hydrochloride. The subsequent refolding proce-dures require excessive dialysis steps and buffer exchangesin high dilutions. (iii) The solubilization of P-CF generatedMP precipitates can already result in functionally folded MPsas shown, e.g., for the multidrug transporter EmrE [5, 35] orfor the human histamine-1 receptor [36]. The successfulrefolding of MPs from inclusion bodies is so far mostly con-fined to outer MPs, whereas the functional refolding of a-helical MPs remains the very exception.

Precipitates obtained from P-CF reactions appear there-fore to consist of less denatured or aggregated MPs if com-pared with inclusion bodies. This assumption is further

supported by the recent observation of folded proteorho-dopsin in P-CF generated precipitates [37]. Structural fea-tures might therefore persist that could support the foldingprocess of distinct MPs upon solubilization. Detergents thatare highly efficient in the solubilization of P-CF generatedprecipitates and that still might allow a functional foldinginclude DPC or the lyso-phosphoglycerols LMPG and LPPG(Table 2). In addition, these detergents provide suitableenvironments for structural analysis of MPs by NMR spec-troscopy [38]. However, the functional solubilization of MPsfrom precipitates is not a general process. Activities of P-CFproduced and solublized samples of the nucleoside trans-porter Tsx and of the human endothelin-B receptor could notbe observed [7, 39].

3.2 CF production of MPs in presence of detergents

(D-CF)

If P-CF expression does not result in functionally foldedprotein, artificial hydrophobic environments could be pro-vided directly into the RM in order to prevent the precipita-tion of the synthesized MPs. Supplementation of detergentsin this D-CF expression mode will result in the formation ofproteomicelles (Fig. 1). The D-CF expression mode offers acompletely new possibility to synthesize MPs. Participationof membranes is excluded and the MPs can interact withdetergents already during or shortly after translation. Theformation of proteomicelles is mainly based on nonspecifichydrophobic interactions and does not require complextranslocation machineries facilitating the insertion of MPsinto membranes. In addition, detergents are availableinstantly at the ribosomes in the RM and no transport ofsynthesized MPs to membranes is necessary. The D-CFexpression mode therefore eliminates problems originatingfrom the transport and translocation processes that oftenrestrict the efficient production of MPs in conventional sys-tems based on living cells [1, 2]. It should also be consideredthat detergent solubilized MPs are needed for structuralapproaches by X-ray crystallography as well as by NMRspectroscopy. Those samples can directly be provided by D-CF expression and the often problematic extraction of MPsout of native membranes with harsh detergents can beavoided.

Not all detergents are suitable for CF expression andsome with a relatively high critical micellar concentration(CMC) like n-octyl-b-D-glucopyranosid (b-OG) or 3-((3-chola-midopropyl)dimethylammonio)-1-propansulfonat (CHAPS)start to inhibit the expression machinery of cell extractsalready at concentrations hardly exceeding their CMCs.However, the majority of commonly used detergents arefound to be tolerated by CF systems in concentrations of atleast several times CMC [7]. Highly suitable is in particularthe group of long chain polyoxyethylene-ethers (Brij-deriva-tives), that can be supplied into CF expression systems atfinal concentrations of more than 1006CMC (Fig. 2A).Other detergents like Triton X-100 or n-dodecyl-b-D-malto-



Table 2. Hydrophobic environments for CF expression

Abbr. Long name Final conc. 6CMCa) Mode Ref.

Detergents

LMPG 1-Myristoyl-2-hydroxy-sn-Glycerol-3-(phosphor-rac-(1-glycerol)) 1% 420 P-CF [7]LPPG 1-Palmitoyl-2-hydroxy-sn-Glycerol-3-(phosphor-rac-(1-glycerol)) 1% n.a. P-CF [7]DPC Dodecylphosphocholine 1% 19 P-CF [7]SDS Sodiumdodecylsulfate 1% 4.2 P-CF [7]Brij-35 Polyoxyethylene-(23)-lauryl-ether 0.1% 10.4 D-CF [7, 53]Brij-58 Polyoxyethylene-(20)-cetyl-ether 1–1.5% 119–178 D-CF [7]Brij-78 Polyoxyethylene-(20)-stearyl-ether 0.5–1% 95–189 D-CF [7]Brij-98 Polyoxyethylene-(20)-oleyl-ether 0.2% 70 D-CF [7]Digitonin Digitonin 0.4% 4.5 D-CF [7, 53]DDM n-Dodecyl-b-D-maltoside 0.1% 15 D-CF [7, 35, 48]Triton X-100 PEG P-1,1,3,3-tetra-methyl-butylphenyl ether 0.2% 13.4 D-CF [7, 8, 53]diC8PC 1,2-Dioctanoyl-sn-glycero-3-phosphocholine 0.1% 8.9 D-CF [7]

Amphipols

C6F-TAC C6F13C2H4-S-poly(tris(hydroxyl methyl)aminomethane) 2–18 mM 6.7–60 D-CF [41]C8F-TAC C8F17C2H4-S-poly(tris(hydroxyl methyl)aminomethane) 2–18 mM 67–600 D-CF [41]HF-TAC C2H5C6F12C2H4-S-poly(tris(hydroxylmethyl)-aminomethane) 2–18 mM 4.5–40 D-CF [41]

Lipids

2 E. coli lipids 0.4% 2 L-CF [5]DMPC 1,2-Dimyristoyl-sn-glycero-3-phosphocholine 0.4% 2 L-CF [15, 44]DOPC 1,2-Dioleyl-sn-glycero-3-phosphocholine 0.4% 2 L-CF [44]DSPC 1,2-Distaeroyl-sn-glycero-3-phosphocholine 0.4% 2 L-CF [44]2 Giant liposomes: phosphatidyl-choline/DOPC/DMPC/cholesterol 5 mM 2 L-CF [45]2 E. coli inner membrane vesicles 0.06–0.08% 2 L-CF [47]

a) Highest reported concentrations.

side (DDM) are also effective for the D-CF expression andsolubilization of MPs, but the presence of higher con-centrations starts to reduce the performance of the expres-sion reaction (Fig. 2B). Further options of detergentsinclude alkyl-glucosides like DDM or steroid derivativeslike digitonin (Table 2). Digitonin and Brij-derivativesappear to be exceptionally efficient for the solubilization ofa diverse variety of MPs in the D-CF expression mode.With both detergent groups, preparative scale amounts ofsolubilized samples of the multidrug transporter EmrE, thenucleoside transporter Tsx, and the rat vasopressin-2receptor could be produced in D-CF reactions [7]. Type andfinal concentration of the supplied detergent can have astrong impact on the quality and the yield of solubilizedMP and these parameters should be subject of optimiza-tion in each D-CF expression approach (Fig. 3) [7, 23, 37].As an example, an initial D-CF expression screen of theinner membrane tyrosine autokinase Wzc of E. coli inpresence of different detergents is illustrated in Fig. 3. Alltested detergents are able to solubilize the synthesized Wzcprotein to some extent at the given concentrations. Thenext step would be to further increase the detergent con-centrations in order to identify the ideal detergent envi-ronment that results in complete solubilization while stillmaintaining high expression levels. The addition of deter-

gent mixtures for the formation of mixed micelles could bea further interesting alternative. Lipids are discussed asimportant structural elements for the folding or stabiliza-tion of certain MPs [40] and providing detergent/lipidcombinations in D-CF reactions could further be con-sidered in order to optimize the quality of the producedMP.

It should be emphasized that extremely mild detergentslike the mentioned Brij-derivatives could increasingly play arole in MP analysis. Those detergents are not able to disin-tegrate membranes and therefore have not been used so farfor the solubilization of MPs by the conventional membraneextraction approach after MP production in cellular expres-sion systems. However, their mildness is now a valuableproperty for their combination with CF synthesis as they arehighly efficient in solubilizing MPs but do not interfere withprotein synthesis. A similar effect might have fluorinated orhemifluorinated surfactants that are strongly hydrophobicbut not lipophilic (Table 2) [41]. These amphipols probably donot compete effectively with protein–protein or protein–lipidinteractions and could therefore stabilize the native fold ofMPs under particularly mild conditions. The mechan-osensitive channel MscL had similar electrophysiological ac-tivity after CF synthesis in the presence of perfluorocarbonsas after expression in vivo [41].



Figure 2. Optimization of detergent concentration for D-CFexpression of MPs. Expression of the nucleoside transporterTsx in E. coli systems in the CECF configuration. The reac-tions were incubated for 12 h at 307C. (A) Increasing con-centrations of Brij-78; (B) increasing concentrations of TritonX-100.

Figure 3. Detergent screen for the D-CF expression of MPs.Expression of the E. coli inner membrane tyrosine autokinaseWzc in E. coli systems in the CECF configuration. The protein wassynthesized in the D-CF mode in presence of various detergentsin the indicated concentrations. The arrow indicates the synthe-sized 79 kDa Wzc protein after electrophoretic separation on a 4–12% SDS-gradient-gel followed by western blotting and immu-nodetection with antibodies directed against a C-terminal poly-(His)6-tag. Molecular masses of marker proteins are given in kDa.B35, Brij-35; B58, Brij-58; B78, Brij-78; B98, Brij-98; DDM; s,supernatant; p, pellet.

3.3 CF production of MPs in presence of membranes

and liposomes (L-CF)

Functional studies of MPs have mostly to be done in membraneenvironments, either in crude native membranes obtainedfrom the expression host cells or in defined artificial proteoli-posomes obtained after reconstitution of purified and solubi-lized MPs. The reconstitution of either P-CF or D-CF producedand solubilized MPs by conventional techniques is feasible andcan be efficient [5, 8, 35, 36]. Alternatively, the direct expressionof MPs into provided liposomes in the L-CF mode can beapproached by providing either microsomal fractions obtainedfrom lysed cells or preformed liposomes composed of definedlipids or lipid mixtures into the RM (Fig. 1). In contrast to pro-teoliposomes obtained after in vitro reconstitution, a prevalentinside out orientation of MPs within lipid vesicles can beexpected since the protein synthesis machinery is only presentoutside of the vesicles. This would significantly facilitate func-tional assays. Lipids and lipid mixtures are tolerated by CF sys-tems at high concentrations and even slightly beneficial effectson the general efficiency of expression could be observed [5].However, problems with the efficient translocation of the syn-thesized MPs might occur like those observed upon expressionin living cells.While the D-CFexpression mode is an establishedtechnique, applications of the L-CF mode for the preparativescale production of MPs are about to emerge and protocols havestill to be optimized. Analytical amounts of functional MPs likeShaker potassium channels or gap junction channel subunitscan be CF produced in presence of microsomal fractions inRRLs [42, 43]. In more recent approaches, functionally foldedbacteriorhodopsin could be produced as membrane integratedprotein after addition of artificial liposomes of defined compo-sition in E. coli extracts (Table 2) [44]. Aplant transporter and themembrane associated protein apo cytochrome b5 could be syn-thesized into preformed liposomes by using WGs [45, 46].However, in all cases again only relatively low amounts of MPcould be synthesized. In a very recent report, two E. coli trans-porters could be CF synthesized into purified E. coli innermembrane vesicles in amounts of several 100 mg/mL of RM[47]. A batch system was used and the activities of the L-CF pro-duced tetracycline pump TetA and the mannitol permease MtlAwere verified by a specific transport assay. The calculated surfacecoverage of the vesicles with up to 40% of recombinant proteinwas significantly higher if compared with data obtained fromcellular expression systems. This work demonstrated that CFexpression of MPs in the L-CF mode can even approach pre-parative scales and this technique might play a significant rolein the next future for the functional analysis of MPs as well as forstudying their specific translocation mechanisms.

4 Applications and quality of CF producedMPs

Commonly asked question for CF systems are: what kind ofMPs can be produced? Are there limitations in protein size or



in the number of transmembrane segments (TMSs)? What isthe quality of CF produced MPs and is CF expression able toproduce functionally folded MPs? What system is best suit-able for the synthesis of eukaryotic MPs? The answers to thosequestions have been addressed in recent times by several in-dependent approaches of individual laboratories. Prokaryoticas well as eukaryotic MPs belonging to a diverse variety offamilies, exceeding 100 kDa in size and with up to 12 TMSshave successfully been CF produced in all three expressionmodes (Table 3). Some reported yields of E. coli systems inCECF configurations are 2–4 mg/mL RM of the transportersTehA, Yfik, and Tsx after P-CF mode expression [5, 7] and 3–6 mg/mL RM of the channel MscL [8] and of the vasopressin-2[7] and histamine H1 receptors [36] after D-CF mode expres-sion, respectively. In a direct comparison of the production of120 different MPs from E. coli either by CF expression or byconventional in vivo expression, a significant higher propor-tion of the targets were synthesized in the CF system and thetwo systems were found to be somehow complementary [48].As a preliminary conclusion, the CF batch configurationappears to be less suitable for the production of proteins ofhigher molecular weight [24, 48]. This might be explained bythe fact that batch reactions are restricted in energy and pre-cursors and shortages obviously begin preferentially to affectthe translation of larger mRNA transcripts. The longer thepolypeptide chain is to be synthesized, the higher is the risk oftranslational pausing followed by premature termination ornuclease attack. Providing sufficient precursors in order tomaintain CF expression reactions for longer time periodscould therefore be an essential prerequisite for the high levelsynthesis of larger MPs, e.g., Wzc (Fig. 3). CFexpression in theCECF or bilayer configuration or more productive batch pro-tocols should be preferred for this application.

The quality of CF produced MPs is much more difficultto assess. Protein quality is a multivalent parameter com-posed of diverse components like purity, homogeneity, fold-ing, or specific activity, that could be addressed by a variety ofbiophysical as well as biochemical approaches. Initial strate-gies for the evaluation of protein quality focus mostly on pu-rity and homogeneity of samples implementing elution pro-file and peak shape analysis after SEC. Well resolved peaks offour P-CF expressed E. coli MPs after gelfiltration of b-OGsolubilized precipitates gave evidence of monodisperse sam-ples [48]. Circular dichroism spectroscopy resulted in spectratypical for a-helical proteins in case of P-CF produced andsolubilized bacterial and eukaryotic transporters [5]. Negativestaining and particle analysis by electron microscopy indi-cated homogenous suspensions of homodimers in case ofseveral G-protein coupled receptors (GPCRs) after D-CFexpression in presence of Brij-78 [49]. Also the homo-dimerization of D-CF produced GPCRs, which in case of thehuman endothelin-B receptor could specifically be attributedto its TMS-1, could be assumed as a good indication forfunctional folding [39]. A further indication for non-aggregated, conformational folded CF produced MPs wereobtained by NMR spectroscopy. Recorded NMR spectra of

purified and 15N-labeled samples of the P-CF produced a-helical tellurite transporter TehA and the cysteine exporterYfik revealed reasonable dispersions most likely originatingfrom homogenously folded proteins [5, 50].

The determination of functional parameters like specifictransport activity or ligand binding kinetics would be a muchclearer indication for the functional folding of CF producedMPs. However, the development of in vitro functional assaysis a laborious and challenging task and for many MPs suchassays either do not exist, or their function or ligands are noteven known. As proofs of principle, some MPs of well devel-oped model systems have been CF produced and analyzed.Substrate specificities and kinetic parameters of the CF pro-duced multidrug transporter EmrE, of the mechanosensitivechannel MscL, and of the eukaryotic ion transporters OCT1,OCT2, and OAT1 matched nicely with previously publisheddata obtained with protein expressed in conventional in vivosystems [5, 8, 35, 51]. In addition, MscL formed its char-acteristic functional pentameric state in detergent micelles.The P-CF produced a-apoprotein of the light harvestingcomplex from Rhodospirillum rubrum could be functionallysolubilized in Triton X-100 micelles and its interaction withthe b-subunit of the complex resulted in specific absorbanceat 820 nm [52]. The functional folding and the correct incor-poration of retinal in CF produced proteorhodopsin whichwas verified by absorbance spectra and photocycle measure-ments [37]. The highest specific transport activity of the b-barrel type nucleoside transporter Tsx was verified after D-CFexpression in presence of Triton X-100 [7]. No activity of Tsxwas detected after P-CF expression followed by solubiliza-tion. In a first report on D-CF expression using WGs, func-tionally folded plant transporters were obtained in presenceof Brij detergents [46].

GPCRs have become in focus for CF expression as thisfamily of MPs is of high interest for the pharmaceuticalindustries and it is supposed to provide the majority of thetargets for modern drugs. The human b2 adrenergic and M2muscarinic acetylcholine receptors as well as the rat neuro-tensin receptor could be synthesized in semipreparativeamounts in batch configurations [53]. Functionality of the D-CF produced b2 adrenergic receptor was demonstrated byradioligand binding-assays with the reconstituted protein. Ina recent report, the human histamine H1 receptor was syn-thesized at high levels in the P-CF configuration [36]. Thefunctional folding of the MP was confirmed by radioassaysafter solubilization in DDM and reconstitution into lipo-somes. Both groups constructed fusions to thioredoxin inorder to improve CF expression of the GPCRs. However,smaller N-terminal tags like the 12 amino acid T7-tag appearto have similar beneficial effects. The human and porcinevasopressin type 2 receptor, the human endothelin B recep-tor and the rat corticotropin releasing factor receptor wereexpressed at high levels in CF expression systems based on E.coli extracts after N-terminal modification with the T7-tag[49]. For the endothelin B receptor, only expression in the D-CF configuration in presence of Brij-detergents resulted in



Table 3. CF expressed MPs

Proteina) Origin Systemb) Quality control Ref.

�20 kDa:

44 MPs (2–6) E. coli EC (P-CF) SEC (4 MPs) [48]MscL (2) E. coli EC (D-CF) Activity, oligomerization [8, 41]EmrE (4) E. coli EC (P-CF) Activity, NMR [5, 7, 55]

EC (D-CF) Oligomerization [35]EC (L/D-CF) Crystallization [15]

SugE (4) E. coli EC (P-CF) Reconstitution, NMR [5]a-apoprotein-LH1 (1) R. rubrum EC (P-CF) Specific absorbance, CD [52]Smr-members (4) Bacteria EC (P-CF) 2 [5, 64]EMP3 (4) Human EC/WG (P-CF) 2 [25]Apo-cytochrome b5 (1) Eukaryotic WG (L-CF) Fusion protein activity [45]

.20 to �30 kDa:

27 MPs (1–7) E. coli EC (P-CF) 2 [48]Bacteriorhodopsin (7) Halobacterium halobium EC (L-CF) Activity [44]

WG (P-CF) Activity [69]Proteorhodopsin (7) Bacteria EC (P/D-CF) Activity [37]ATP6V0B (5), SSR2 (7) Human EC/WG (P-CF) 2 [25]OEP24 (b-barrel) Eukaryotic EC (L-CF) Activity [6]

.30 to �50 kDa:

Tsx (b-barrel) E. coli EC (D-CF) Activity [7]pOmpA (?) E. coli PURE (L-CF) Protease protection [70]FtsQ (1) E. coli PURE (L-CF) Protease protection [70]TehA (10) E. coli EC (P-CF) Reconstitution, NMR [5]NhaA (10), MelB (12), UbiA (8) E. coli EC (P-CF) 2 [3]HMOX2 (1), ATP1B1 (1), PXMP3

(2), PL6 (7)Human EC/WG (P-CF) 2 [25]

AtPPT1 (6), AtTPT (9) A. thalianaOsPPT1–3 (?) O. sativa WG (D/L-CF) Activity [46]V2R (7) Rat EC (P/D-CF) 2 [7]b2AR (7), M2R (7), NTR (7) Human EC (P/D-CF) Activity (b2AR) [53]ETB (7), V2R (7), CRF (7) Activity (ETB) [39]MTN1B (7), NPY4 (7) Human/rat EC (P/C-CF) Monodispersity [49]ETB (7) Human EC (D-CF) Oligomerization, activity [39]H1R (7) Human EC (P-CF) Activity [36]Connexins (4) Human/rat RRL (L-CF) Oligomerization, activity [42]AQP (6) N. tabacum EC (P-CF) 2 [3]

.50 kDa:

MtlA (8) E. coli PURE (L-CF) Protease protection [70]E. coli EC (L-CF) Activity [47]

TetA (12) E. coli EC (L-CF) Activity [47]OCT1/2 (12), OAT1 (12) Rat EC (P-CF) Oligomerization, activity [51]MLSTD1 (2), TLR1 (1) Human EC (P-CF) 2 [25]Shaker H4 Eukaryotic RRL (L-CF) Activity [43]

a) Proposed number of TMSs is given in parenthesis.b) Proteins were produced either in batch or CECF configurations, the applied expression modes are indicated.

EC, E. coli system; RRL, rabbit reticolocyte lysate system; WG, wheat germ system.

homogenous protein samples with ligand binding activity,whereas solubilized precipitates obtained after P-CF expres-sion remained aggregated.

It is not possible to predict which system and whichexpression mode might be most promising for the CF pro-duction of a given MP target. E. coli systems have been

shown to be highly productive for the synthesis of prokar-yotic as well as of eukaryotic MPs in high quality. Dataobtained with WGs are still very limited, but there is nodoubt that also wheat germ extracts will be highly suitable forthe efficient production of MPs. However, the question is ifand when it might be an advantage to prefer CF expression of



MPs in WGs instead of using E. coli systems. The prepara-tion of wheat germ extracts is much more critical and it takessignificantly longer if compared with the preparation of E.coli extracts. WGs might provide a more genuine folding en-vironment for eukaryotic MPs. The rate of peptide growth oneukaryotic ribosomes in vivo is five to ten times slower ifcompared with prokaryotes, thus promoting the cotransla-tional folding of proteins. This could be in particular advan-tageous for the production of multidomain proteins, as itreduces the risk of nonproductive contacts between unfoldedregions. However, in bacterial CF expression systems theprotein synthesis rate appears to be reduced and it thus couldbecome comparable or even lower to that measured ineukaryotic cells [54]. A further proposed benefit could be thepotential of PTMs in wheat germ extracts. However, the effi-cient modification of MPs would require the supplementa-tion of microsomal fractions which in principal could also beadded into E. coli extracts. A first attempt to compare the CFexpression of eukaryotic proteins including also some MPsin E. coli and wheat extracts indicated some better perfor-mance of the WG [25]. However, different configurations andstrategies have been used that could have been contributed tothis preliminary conclusion. It should finally be realized thatthe expression mode can have a significant impact on thequality of a synthesized MP, while all three expressionmodes can principally result into functional MPs (Table 3).Some MPs like the multidrug transporter EmrE can berecovered as folded protein after expression in the P-CF [5,55], D-CF [35], as well as L-CF modes [15].

5 CF expression of MPs in throughput andproteomics applications

CF expression systems reduce the complexity of MP pro-duction essentially to the transcription/translation process.Success rates in throughput expression screens are there-fore higher as in conventional in vivo expression systems.In addition, a number of critical and often highly variableparameters like efficient gene delivery, template stability incells, suppression of background expression, and inductionof expression as well as considerations about optimal cellphysiologies are eliminated. Furthermore, samples can betaken at any time point without disrupting the reaction.The possibility to use linear DNA fragments as templatefor CF expression is a further benefit for throughputapplications. Products from appropriately designed PCRreactions including suitable promoter sequences could beused directly for expression analysis and time consumingsubcloning procedures are not necessary [56, 57]. Besidesscreening the expression of a larger number of differenttargets at similar conditions, the open nature and easyaccess of CF reactions further allows the screening of ahigh number of compounds and additives in order todevelop optimal expression protocols for few but highlyimportant targets. The possibility of this throughput

expression condition screening enables the design of indi-vidualized CF expression protocols for each new MP target.This feature is specific for CF systems as the closed char-acter of cellular expression reactions determined by the cellmembrane usually confines screening options to only afew parameters like choice of vectors and host strains orvariation of incubation conditions. Specific properties andrequirements of individual MP targets can now be con-sidered for the development of CF expression protocols byaddition of target specific beneficial compounds. If suchindividual care is taken, the success rate of CF MP expres-sion is considerably higher than in any other expressionsystem. Throughput strategies are typical applications forautomatic processes and a combination of selected param-eters could be analyzed in one screening level as illustratedin a schematic process in Fig. 4. Iterative screening reac-tions combined with corresponding feedbacks from theevaluation level to the basic master protocol could finallydefine optimized reaction protocols for each specific MPtarget.

CF systems are established tools for functional and struc-tural proteomics with soluble cytoplasmic proteins [58–60].Automated processes based on CF expression technologies arealready routine techniques for structural proteomics by NMRspectroscopy [61]. Functional proteomics will increasinglyprofit in the near future from protein arrays generated by CFsynthesis. The high flexibility of CF expression systems for theproduction of folded proteins in combination with the parallelon-chip synthesis of protein arrays directly from PCR tem-plates will considerably facilitate throughput approaches forprofiling, interaction, and screening purposes. The versatilityof CF systems allows the rapid expression of cloned oruncloned DNA sequences concomitant with the instant array-ing of the produced proteins, domains, or peptides by specifictrapping techniques [62]. A recently developed microfluidicarray device further increases the performance of thisthroughput application as the batch configuration can bereplaced by a modified CECFconfiguration [63]. For MPs, thoseapplications are just about to emerge. The high success rate offirst throughput CF expression screens including MPs willprobably further accelerate this progress [25, 48]. Proteomictechniques for the analysis of MPs often suffer from low pro-duction yields that do not reach the LODs of typically employedtechniques like MS. Higher expression rates as well as theabsence of lipids might facilitate proteomics studies with CFproduced MPs. In a recent report, most of the TMSs of the CFproduced histamine H1 receptor could be covered by MS anal-ysis, indicating a good potential for MP profiling purposes [36].

6 Structural approaches with CFproduced MPs

Despite CF expression of MPs is a very new technique,already a first 3-D structure of a multidrug transporter couldbe solved with participation of CF produced samples [15].



Figure 4. Throughput process for the protocol optimization of CF expression. (A) Scheme and sequence of the reaction process. (B) Opti-mization of two reaction parameters in batch configuration in microplate formats. (C) Result of one optimization process and identificationof optimal concentrations for the ions Mg21 and K1.

[35S]-Met labeled EmrE was synthesized in an E. coli CF sys-tem in batch configuration. Crystallization conditions for theCF expressed EmrE were similar to those found for proteinobtained from in vivo expression systems. This coincideswith the previously observed similarity of functional proper-ties of CF and in vivo expressed EmrE [5, 35, 64]. Individualamino acid types or selected combinations can easily andcompletely be replaced in CF reactions by labeled derivatives.This guarantees efficient label incorporation into the syn-thesized proteins. In contrast to conventional labeling tech-niques with cellular expression systems, the backgroundlabeling is minimized as no other proteins besides the MPtarget are synthesized in CF reactions in significantamounts. This feature is of high value for structural ap-proaches by X-ray crystallography and even more for NMRspectroscopy. The fast production of MPs in combinationwith their efficient labeling with the stable isotopes 13C, 15N,and/or 2H by CF expression enables a variety of new struc-tural approaches by NMR. The small volumes reduce costsfor amino acid specifically labeled samples tremendously. Inaddition, the absence of background labeling allows even theanalysis of labeled MPs in rather impure samples [65, 66].

The complete labeling of MPs often leads to very crowdedspectra with massive signal overlaps. More simplified spectracan be obtained by amino acid specific or combinatoriallabeling strategies, which in addition are valuable tools for

the assignment of larger proteins or for the development ofautomated throughput assignments in structural proteomicsprojects [65, 67]. Combinatorial labeling schemes enabledthe assignment of a derivative containing seven TMSs of thebacterial tellurite transporter TehA [50]. The reduced overlapin the specifically labeled samples could be exploited in orderto obtain sequential connectivities that could not be extractedfrom uniformly labeled samples due to severe peak overlap.Combinatorial labeling is an ideal application for CF expres-sion. Besides the replacement of the desired natural aminoacids with their labeled derivatives, no other changes in theexpression protocol have to be done. The expression rates donot change and NMR ready samples of combinatorial labeledMPs in any amino acid combination can be obtained in only2 days. Specific or even combinatorial labeling of MPs is onthe other hand, very difficult to perform in cell-basedexpression systems. Besides lower efficiencies of MPexpression, problems with isotopic scrambling caused byamino acid metabolism, inefficient label incorporation, andthe requirement of multiple auxotrophic strains usually pre-vent such approaches.

The routine analysis of MP structures by NMR still has toovercome significant problems. Increased rotation–correla-tion times, which directly depend on the size of the analyzedprotein, result in massive line broadening in NMR spectra.Partial or complete overlaps or even vanished signals are the



consequences. Folded MPs require a hydrophobic environ-ment consisting of micelles or liposomes that furtherincrease the size of the rotating protein complex. Only fewdetergents like LMPG or LPPG still result in acceptablespectra and appear therefore to be suitable for NMR meas-urements of MPs [38]. Additionally, fold similarities andtypically strongly biased amino acid composition of MPsresult in similar chemical shifts in protein backbone selec-tive NMR spectra. Distance dependent nuclear overhausereffect (NOE) or angle-constrains mainly based on the dis-persion of a-carbon and a-proton chemical shifts are com-monly used to determine final 3-D folds of proteins. Thoseconstrains are not efficient for a consistent determination ofMP structures, since their NH-based 3-D spectra providesonly few and hard to extract long range NOEs. However, four-dimensional N,N-linked experiments reduce spectral over-laps of overall a-helical proteins and proposed secondarystructure elements could be verified with the observed NOEsin such a way that a global fold of an MP could be defined.Using the method of paramagnetic relaxation enhancementto further estimate interhelical distances within the MP fold,additional constrains for NMR based structure determina-tions could be obtained [68].

7 Perspectives

The high success rate in expression screens and their con-siderable efficiency in preparative scale MP production openCF systems a wide perspective of applications. The potentialsof the new strategies to synthesize MPs directly into deter-gents or into provided liposomes have by far not completelyexplored yet. The efficiencies of membrane insertion in theL-CF expression mode will further be optimized, e.g., byaddition of hydrophobic tags to MPs or by providing essen-tial compounds of translocation systems into the reaction.Providing mixtures of detergents and lipids might become afurther option to improve the stability and quality of thesynthesized MPs. The contact of MPs with harsh detergentscan now be completely avoided during preparation protocolswhich could enable CF expression approaches with also lessstable MPs or MP complexes. In general, the intrinsic prop-erties of the selected CF expression system, like extractsource, expression mode, and configuration of the reactionmust be considered for best performance. Further improve-ments of CF reaction protocols for specific application pur-poses like the cotranslational incorporation of artificialcofactors or the single site labeling of proteins with tRNAscarrying chemically conjugated amino acids such as isotopic,fluorescent, or biotinylated derivatives by the stop codonsuppression methodology, are expected developments of thenear future. Furthermore, CF expression systems based onan extended variety of extract sources will be established. CFexpression systems offer clear benefits in acceleratingthroughput and proteomics applications as the batch orbilayer configurations are easy to set up and reactions are fast

and with sufficient efficiencies to isolate micrograms of pro-tein. However, for applications such as structural analysis,where higher amounts of protein are needed, the optimiza-tion of protocols with the more productive CECF configura-tion is often necessary.

A potential problem is that no protein quality controlmechanisms are present in CF expression systems. In cells,denatured or partly misfolded MPs are usually not trans-ported or translocated into membranes. Misfolded proteinparticles might therefore also end up in micelles or liposomesin CF expression systems. However, the so far documentedquality of CF produced transporters and GPCRs is promisingthat even crystallization grade MP samples can be obtained.In standard CF systems without added microsomes, PTMs ofMPs such as glycosylations are absent, which could evenresult in an improved homogeneity of MP samples.

The authors have declared no conflict of interest.

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production of membrane proteins using cell-free expression systems

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