Membrane protein production in Escherichia coli cell-free lysates

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Review

Membrane protein production in Escherichia coli cell-free lysates

Erik Henrich, Christopher Hein, Volker Dötsch, Frank Bernhard

⇑

Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W. Goethe-University, Frankfurt-am-Main, Germany

a r t i c l e

i n f o

Article history: Received 16 March 2015 Revised 17 April 2015 Accepted 21 April 2015 Available online 1 May 2015 Edited by Wilhelm Just Keywords:

Synthetic biology Nanodiscs

Membrane protein solubilization Expression modes

Escherichia coli lysates

a b s t r a c t

Cell-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 conven-tional 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 stabiliza-tion of membrane proteins can rastabiliza-tionally 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 strate-gies for the successful production of high quality membrane protein samples.

1. Introduction

Membrane proteins are one of the most important protein classes and they represent the majority of targets for modern drugs. Numerous limitations in their production by conventional in vivo expression systems largely retarded their functional and structural characterization. Toxicity, limited membrane space for their functional folding as well as inefficient transport and mem-brane insertion mechanisms pose significant barriers. Synthetic cell-free (CF) expression systems eliminate most of these classical problems in membrane protein production and thus rapidly became a new platform for their characterization[1–5]. In addi-tion, CF expression unifies a number of further beneficial proper-ties such as non-complicated operation, high success rates and large flexibility for the fast development of an array of diverse applications[6].

CF expression is a relatively old technique and known since more than 50 years after publication of the famous experiments of Nirenberg and co-workers on deciphering the genetic code with Escherichia coli lysates[7]. While the technique has been in use for decades as analytical scale protein production system, the overall synthesis efﬁciency was considerably improved after developing reﬁned protocols and reaction designs [8,9]. Based on its easy

access and excellent advantages for the efﬁcient labeling of proteins, CF expression systems became routine platforms for the production of soluble proteins for structural analysis[10–12]. In recent times, the variation of CF systems rapidly expanded and lysates of various organisms have been analyzed for their efﬁcien-cies in protein production.

The available CF systems show tremendous differences in view of efﬁciencies and applications and selection of appropriate CF lysate sources can be crucial for the subsequent performance of a project. At the current state of the art, lysates from E. coli have been successfully used for the production of the by far highest variety of membrane proteins with regard to size, topology, function and ori-gin[13,14]. Documented applications include functional studies, X-ray crystallography, nuclear magnetic resonance (NMR) struc-tural analysis as well as the characterization of large membrane protein assemblies[15–18]. Key advantage of E. coli lysates is their reliable efﬁciency in protein synthesis allowing the routine pro-duction of even large membrane proteins in mg amounts per one mL of reaction. Detailed protocols for the preparation of E. coli lysates and for the set-up of corresponding CF reactions have been described[19–22]. CF membrane protein production was initiated approximately 10 years ago and applications are continuously expanding (Fig. 1). In parallel, the number of system variations and protocols is still increasing. The intention of this review is to give a guideline on what kind of decisions and considerations have to be made when approaching the CF expression of a membrane protein in E. coli lysates. Complementary information summarizing current approaches can be obtained in several recent reviews[1,3–

http://dx.doi.org/10.1016/j.febslet.2015.04.045

Biomolecular Magnetic Resonance, J.W. Goethe-University, Max-von-Laue Str. 9, Frankfurt-am-Main 60438, Germany. Fax: +49 69 798 29632.

E-mail address:fbern@bpc.uni-frankfurt.de(F. Bernhard).

FEBS Letters 589 (2015) 1713–1722

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5,23]. E. coli lysates are used as coupled transcription/translation systems with linear or circular DNA as template. Transcription is usually controlled by supplied T7 RNA polymerase which can be isolated out of overexpressing strains according to routine proto-cols[24]. Alternatively, E. coli lysates with already adjusted con-centrations of T7 RNA polymerase are commercially available. Expression controlled by endogenous E. coli RNA polymerases could further be efficient[15,25]. Protocols are developed and opti-mized in small volumes of microplates and subsequent scaling up to even multiple liter volumes is possible[26]. Expression efficien-cies close to one mg per mL are already possible with single com-partment batch reactions and by using E. coli lysates from optimized strains as well as specific reaction conditions[27–29]. Crude E. coli lysates can even be replaced by reconstituted transla-tion machineries composed out of the purified individual compo-nents[30].

Protein production efficiencies considerably increase by provid-ing additional reservoirs of small molecule precursors in a second compartment[9]. Protein synthesis in such continuous exchange cell-free (CECF) configurations is ongoing for many hours. Container geometry, the ratios of reaction and reservoir volume as well as the exchange surface in between the two compartments are important parameters that determine the final protein produc-tion efficiencies. A number of alternative opproduc-tions comprising indi-vidual as well as commercially available CECF containers have been proposed[22]. In addition, recently developed dialyzer car-tridges in combination with standard deep-well microplates are well suitable for analytical and semi-preparative scale screening reactions in the CECF configuration.

2. Project strategies: approaching CF production of membrane proteins in E. coli lysates

CF expression can generally be considered as a complementary approach to expression in E. coli cells in cases when the cellular expression fails or only minute amounts of protein are synthesized

[13]. In addition it is a good choice for the quick labeling of a pro-tein as DNA templates can easily be shuttled in between expres-sion systems with E. coli cells or CF lysates. Initial considerations when approaching CF expression should address (i) whether the technique is generally appropriate for the production of the selected target, (ii) which lysate should be used, (iii) which reac-tion environments should be provided and (iv) how sample quality can be controlled and evaluated (Fig. 2). Completely integral mem-brane proteins are preferable and targets containing up to 12 transmembrane segments and exceeding 100kDa have been syn-thesized so far[14,31]. In contrast, membrane proteins containing large soluble domains such as anchored or single span membrane

proteins are more likely to experience folding problems during CF expression. Supplied detergents or other artiﬁcial hydrophobic compounds may affect the proper folding of the soluble domains. The presence of multiple disulﬁde bridges could furthermore cause problems and the screening of redox systems should then be con-sidered[32].

Common lysate sources are the E. coli K strain A19 or BL21 derivatives such as the strains C41 or C43 known as the gold stan-dards for membrane protein production in vivo[33]. In addition to different strains, also several modiﬁcations of lysate preparation can be considered. Standard S30 lysates (i.e. fractionated by cen-trifugation at 30 000g) still contain residual cellular membrane fragments in amounts of up to approximately 100

l

g lipids per one ml of lysate[18,34]. Inserted endogenous porins or ion chan-nels may thus cause background signals when working with highly sensitive detection methods such as electrophysiological measure-ments. This background can be avoided by using S60 extracts pre-pared at 60 000g[34]. On the other hand, S12 extracts prepared at 12 000g can be used alternatively to S30 extracts for other targets

[35]. Protein labeling might be performed in lysates of speciﬁcally designed E. coli knock out mutants or in chemically pre-treated lysates[36]. Lysates could further become enriched for chaperones that might improve the functional folding of synthesized proteins

[37]. Chaperone induction upon lysate production or supply of chaperones could alter the resulting lysate proteomes into more favorable environments.

CF expression is a highly versatile synthetic biology approach. Initial reaction designs can therefore be adjusted according to the individual requirements of a membrane protein or to the intended application (Fig. 2). Samples suitable for structural approaches can be obtained by the co- or post-translational solubilization of CF expressed membrane proteins in a number of detergents. Numerous membrane proteins strictly require lipids in order to support folding and stability[38,39]. Expression in presence of pre-formed nanodiscs or with supplied detergent micelles containing particular lipids will therefore be a good choice. The multitude of options in order to manipulate reaction conditions will usually generate a comprehensive library of samples containing the target protein in quite different environments and probably also with sig-nificant variations in quality (Fig. 3). The strategy resembles the classical ‘‘funnel’’ model of expression screens for membrane pro-tein crystallization[40,41]. In cellular expression systems, large target libraries are screened and selection by expression efficien-cies, membrane insertion and isolation procedures usually results into a subset of few promising targets (Fig. 3). In contrast, a num-ber of significant differences do exist with the CF-funnel model. First, the high success rate of CF expression allows focusing on few selected targets that are of prime interest. Second, a large sam-ple library of the desired target solubilized in a variety of different environments is generated. Third and most important is that screening tools mainly comprise additives affecting sample quality such as folding environments composed out of various combina-tions of hydrophobic compounds, stabilizers and ligands rather than parameters affecting production efficiencies as in the classical cellular funnel. The result of the CF-funnel model is therefore a subset of samples having the desired and functionally folded target in different environments such as detergent micelles or nanodiscs (Fig. 3).

The functional folding of a membrane protein usually shows more or less clear preferences for particular CF reaction environ-ments[39,42]. Efficient sample quality evaluation tools preferen-tially applicable directly in the crude CF reaction mixture such as specific ligand binding assays or enzymatic activity measurements are therefore valuable. The availability of such tools is frequently a major bottleneck in CF expression projects as only more advanced and time consuming assays such as size exclusion profiling or Fig. 1. PubMed referenced reports on the CF expression of membrane proteins in

between 2004 and 2015.

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Fig. 2. Basic considerations important for the initial design of CF expression processes. Depending on the target properties and on the intended application, individual options and modules will be selected for the design of the ﬁnal CF expression strategy.

Fig. 3. Schematic comparison of cellular versus CF screening strategies for membrane protein production. Both processes are compared as a ‘‘funnel’’ model. Symbols represent the individual membrane protein targets. The main differences in between the two strategies exist in the target list and in the screening strategies as discussed in the text. The CF funnel starts with one desired target represented by the star and generates a library of samples in a variety of different environments comprising hydrophobic solubilizing compounds, stabilizers or ligands. In contrast, the cellular funnel starts with a larger list of different targets that will all be synthesized in identical or very similar conditions. Quality control e.g. implementing functional assays will select a sample subset with the identical target solubilized in different environments in the cell-free funnel, whereas a subset of different targets solubilized in identical environments are usually selected in the cellular funnel.

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analysis of transport function are available. However, an initial short list of promising expression conditions can be determined by using fusions with monitors such as green ﬂuorescent protein (GFP) (Fig. 4) [43]. Reaction conditions resulting into aggregated membrane proteins could then instantly be identiﬁed by using fusions with C-terminally attached GFP derivatives [44,45]. Furthermore, concentration screens of compounds affecting yield and solubilization of a membrane protein can rapidly be monitored.

3. The ﬁrst milestone: improving CF production efﬁciencies of membrane proteins

CF expression reactions have a largely reduced complexity if compared with living cells as environment for protein overproduc-tion. Cytotoxic effects are eliminated and the lysate preparation procedure enriches compounds relevant for the expression machinery. The open access furthermore facilitates the addition of e.g. protease inhibitors in order to prevent the degradation of synthesized proteins. As a result, expression problems such as low yields are usually only associated with inefﬁcient translation and can rapidly be addressed by few standardized optimization steps. Adjusting the Mg2+_{ion concentration for each DNA template}

is always mandatory as a ﬁrst trouble shooting step and it has a clear impact on the ﬁnal expression success[46,47]. Target frag-mentation could be caused by premature termination of transla-tion and may be addressed by using codon optimized synthetic genes.

The initial DNA template design usually comprises a number of alternative options that are either used for protocol development or for final preparative scale expressions (Fig. 4). Inefficient initia-tion of translainitia-tion is the most frequently encountered bottleneck when approaching high yield CF expression. Prime determinants of translation initiation are the first few codons of the mRNA and suboptimal nucleotide sequences could restrict their access by ribo-somal RNA subunits[48]. A rapid pragmatic approach in order to improve expression yields is the attachment of short sequence opti-mized N-terminal expression tags (Fig. 4)[49–51]. Screening a low number of nucleotide-optimized expression tags mostly results into significantly improved expression efficiencies within short time. If the modification of the target protein with the few amino acids of the expression tag causes non-desired altered protein characteris-tics, enrichment of the first six to seven natural mRNA codons for A or T nucleotides by directed mutagenesis will be an alternative strategy as demonstrated with a membrane-integrated domain of an ABC transporter[52]. Additional tags useful for rapid purification

(i.e. poly(His)n-tags or Strep-tags) could be attached to the

C-terminus of the target protein (Fig. 4).

Further major determinants for the expression yield are (i) the selected conﬁguration, i.e. batch versus CECF, (ii) the selected energy resources and precursor concentrations and (iii) the precur-sor reservoir volume in the CECF conﬁguration as well as its poten-tial refreshing during the reaction. Yields of even identical targets from different reports are therefore hard to compare as usually quite different reaction conditions have been used. Examples of detailed protocols and cost calculations for the preparative scale production of membrane proteins intended for structural projects have been published and could be taken as preliminary bench-marks[18,22].

4. Reﬁning sample quality: designing synthetic environments with additives

If the above mentioned optimization strategies are considered, the success rate of membrane protein production in E. coli lysates is very high and independent from membrane protein topology or function. Based on the established production protocol, the sub-sequent reﬁnement procedure will focus on the systematic screen-ing of expression environments suitable to support the functional folding and stability of the synthesized membrane protein (Fig. 5). Three distinct expression modes can serve as starting points for sample quality optimization. In the precipitate forming (P-CF) mode, the synthesized membrane proteins precipitate after translation and are solubilized post-translationally in detergent micelles. In contrast, in the detergent-based (D-CF) and the lipid-based (L-CF) modes, the synthesized membrane proteins will be co-translationally solubilized. Pure detergents, mixed micelles, micelles containing lipids, surfactants or amphipols could be sup-plied in the D-CF mode, while in the L-CF mode lipid bilayers are provided[3,53–58]. Overviews on the different types of artiﬁcial hydrophobic environments including their particular properties are given elsewhere[3,59].

Co-translational solubilization in either D-CF mode or L-CF mode has an obvious impact on the translation kinetics of mem-brane proteins. Whereas the synthesis of soluble proteins such as GFP is not affected by the presence of supplied detergents or lipids, the synthesis rate of membrane proteins is often notably reduced

[45]. Higher yields of membrane proteins can thus be obtained in the P-CF mode without supplied hydrophobic compounds. The pre-cipitates may retain folded structures and excessive refolding pro-tocols are usually not applied for their subsequent solubilization

[60]. Depending on the membrane protein, a number of detergents

Fig. 4. Improving the CF expression yield of a membrane protein target. The basic system efficiency, e.g. the E. coli lysate quality, is monitored by GFP expression benchmarking. Adjusting the Mg2+_{ion concentration for each target DNA template is furthermore crucial. Expression depends on efficient initiation of translation and can be} optimized by either addition of small expression tags or by appropriate silent mutagenesis of the first natural 5’codons. Additional optional modifications include the attachment of C-terminal monitors such as GFP and of small standard purification-tags.

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such as lyso-phosphoglycerol derivatives, phosphocholines or DDM are efficient in solubilization. The P-CF synthesized MraY translocase of Bacillus subtilis showed the highest specific activity after solubilization in DDM[46]. Further functional or even high resolution structural characterizations of P-CF generated mem-brane protein samples have been exemplified[15,31,47,61–64].

However, some membrane proteins may unfold irreversibly in the P-CF mode[46]. The next step would thus be to switch to the D-CF mode (Fig. 5). Systematic screens of detergents or surfactants with deﬁned working concentrations that are tolerated by the CF system have been published[3,65–69]. Most commonly used are long chain Brij derivatives as well as peptide surfactants, digitonin, the micelle forming short chain lipid di-heptanoyl-phosphocholine or ﬂuorinated surfactants[16,61,69,70–74]. Suitable environments are still hard to predict. The Agrobacterium curdlan synthase was active after D-CF expression in peptide surfactants but not in Brij derivatives [75], whereas numerous olfactory receptors showed similar activity in peptide surfactants or in Brij detergents

[67,74]. High quality samples of the mitochondrial carrier Ucp1 were obtained with ﬂuorinated surfactants[69]. Screening mixed micelles or micelles containing some lipids for better stability of membrane proteins could further be advantageous [69]. The NMR structure of proteorhodopsin as well as the crystal structure of the eukaryotic Acetabularia rhodopsin II were obtained after their D-CF expression in E. coli lysates in presence of digitonin micelles containing some phosphocholines[16,17].

Membrane protein folding upon co-translational solubilization can further be supported by providing stabilizing additives. Addition of the all-trans retinal cofactor is required for the

functional folding of rhodopsin derivatives [16,17,76]. The con-trolled co-expression of individual subunits enabled and stabilized the assembly of functional ATP synthase [15,77]. Redox systems such as combinations of oxidized and reduced glutathione could be beneficial for the formation of disulfide bridges in eukaryotic membrane proteins. Tightly binding small molecule ligands could further help to support conformational stability e.g. during CF expression G protein-coupled receptors (GPCRs) as already shown in crystallization studies[78,79]. Efficient strategies for extensive post-translational modifications of membrane proteins in E. coli lysates have so far not been developed and are still future perspec-tives. The addition or co-expression of corresponding modifying enzymes such as glycosyl transferases generally appears to be fea-sible but still awaits further approval[80].

5. Implementing bilayers: production of lipid dependent membrane proteins

Lipids are the natural environment of membrane proteins and are sometimes essential as stabilizing or even structural elements

[38,81,82]. In addition, some membrane proteins do not fold in presence of detergents or do not even tolerate any contact with detergents [46]. The L-CF mode implementing supplied mem-branes as preformed liposomes, nanodiscs or as isolated micro-somes is mandatory for these types of proteins (Fig. 5). However, membrane protein insertion into bilayers is a different and less efﬁcient process if compared with their solubilization in deter-gents. Natural insertion machineries could be used by providing Fig. 5. Sample quality evaluation and reﬁnement of CF expression conditions. The three basic modes P-CF, D-CF and L-CF can be implemented either simultaneously or sequentially. For each mode, numerous combinations of additives will be supplied and the quality of the resulting samples will be screened in appropriate assays.

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microsome fractions or by the directed synthesis of translocons

[2,83,84]. However, supplied artificial bilayers are devoid of any proteins which is in contrast to the typically high protein to lipid ratio of cellular membranes. This characteristic is most likely advantageous for spontaneous insertion of membrane proteins as artificial bilayers are then better accessible and provide more space for insertion [34,71,85–88]. In addition, the specific topology of artificial bilayers, e.g. the two-side access and the interface in between membrane and membrane scaffold protein in nanodiscs can further support membrane protein insertion. Providing some detergent together with liposomes may further weaken bilayers and promote insertion[89].

Well deﬁned and preformed bilayers can be added into CF reac-tions either as liposomes or as nanodiscs (Fig. 6). In particular the nanodisc technology provides excellent synergies to CF expression

[90,91]. Nanodiscs and liposomes of identical membrane composi-tions differ signiﬁcantly in a number of properties. Nanodiscs are stable and can be supplied in ﬁnal concentrations of at least 100

l

M giving stoichiometric ratios even with highly expressing membrane proteins. In contrast, pre-sized liposomes tend to fuse during the reaction and might even precipitate [45]. Furthermore, nanodisc membranes and liposomes will have quite different topologies with regard to membrane curvature, lateral pressure and accessibility. Insertion of L-CF expressed GPCRs and other membrane proteins was in particular efﬁcient with nan-odiscs [42,76,92,93]. L-CF produced nanodisc complexes with membrane proteins are highly soluble and suitable for many appli-cations such as ligand binding studies or the characterization of membrane protein oligomerization[42,94,95].

The selection of suitable membrane compositions is a crucial point and lipid size, charge and resulting membrane topology are important parameters for the membrane insertion and folding efﬁ-ciency of membrane proteins (Fig. 6). The denaturation tempera-ture of bacteriorhodopsin was shown to change signiﬁcantly with the membrane composition[96]. Furthermore, the sterol content of liposomes modulated the water permeability of inserted aqua-porins[97]. Transition temperatures of the designed membranes should always be below the usual CF reaction temperatures in between 25 °C and 35 °C in order to facilitate protein insertion

[98]. Lateral pressure in the membrane depends on the lipid head-groups as well as on the alkyl chain geometries and it is increased by double bonds in cis conformation[99,100]. Spontaneous inser-tion of membrane proteins into lipid bilayers is higher at low

membrane elastic stress. Consequently, phosphocholine lipids or even single chain lyso-phosphocholine lipids that reduce the cur-vature stress could be more successful in promoting membrane protein insertion in L-CF expression approaches. Negatively charged headgroups of phosphoglycerol derivatives could further improve membrane insertion and folding efﬁciency [101]. Accordingly, the functional folding of E. coli MraY translocase enzymes responsible for bacterial cell wall precursor formation was only observed after L-CF expression with nanodiscs assembled with lipids containing phosphoglycerol headgroups[39].

6. The core application: functional studies with CF expressed membrane proteins

Applications for the functional characterization of CF expressed membrane proteins appear to be hardly limited and already cover numerous examples of enzymes, pores, channels, transporters, receptors as well as of fully assembled multi protein complexes

[1,5]. However, despite the common use of E. coli lysates, the indi-vidual protocols for expression and processing differ signiﬁcantly in view of the implemented expression mode, of the provided hydrophobic environments and of the selection of supplemented compounds. While high quality samples have been prepared out of all three expression modes, the preferred selection strictly depends on the requirements of the individual membrane protein targets. In general, the P-CF and D-CF modes are more interesting for structural studies, whereas the L-CF mode in combination with liposomes or nanodiscs becomes important for the characteriza-tion of lipid dependent activities of membrane proteins

[39,42,76,92,93].

Most current data are obtained from the characterization of CF expressed rhodopsin derivatives, GPCRs, porins and channels as well as of membrane integrated enzymes. Underlying reasons for this selection bias are the availability of fast or highly sensitive functional assays for these classes of membrane proteins such as the folding dependent light absorption of rhodopsin derivatives or the speciﬁc and high afﬁnity ligand binding of GPCRs. Those assays are essential for the screening of suitable expression condi-tions in a reasonable period as pointed out in the previous chap-ters. A large variety of GPCRs synthesized in E. coli lysates have been characterized. Most GPCRs have been D-CF expressed in pres-ence of detergents[74,102–105], peptide surfactants[55,67,106]

or amphipol like substances[68]. Alternatively, P-CF expression

Fig. 6. Important lipid characteristics for the insertion and stabilization of membrane proteins exempliﬁed on the basis of a nanodisc complex. Lipid properties such as type of headgroup, the length of the alkyl chain or saturation affect the ﬂuidity, thickness, charge and lateral pressure of the bilayer. Direct lipid interaction with membrane proteins can have a further impact on protein folding, stabilization or activity.

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with subsequent detergent solubilization was implemented

[103,107]. Even synthetic membranes built by diblock copolymers could become a suitable expression environment for GPCRs[108]. Recent studies with the human endothelin receptors indicated that L-CF expression into preformed nanodiscs could be a fast tool for the subsequent characterization of GPCR/ligand interactions by surface plasmon resonance techniques even in crude CF reaction mixtures[42]. Alternatively, the direct insertion of CF expressed GPCRs as well as of other membrane proteins into tethered lipid bilayer membranes immobilized on chip surfaces could enable future drug screening attempts[109–111].

7. Arising perspectives: structural studies with membrane proteins

High success rates, rapid access to protein samples and efﬁcient productivities make CF expression systems with E. coli lysates suit-able for structural studies[112]. Seleno-methionine labeled sam-ples of the multidrug transporter EmrE were a ﬁrst example of a CF synthesized and crystallized membrane protein [113]. Only low diffracting crystals were obtained with a human voltage-dependent anion channel[114], whereas high resolution crystal structures of D-CF expressed Acetabularia rhodopsin II

[17] and the P-CF expressed bacterial diacylglycerokinase DgkA

[18]were solved at 3.2 Å and at 2.28 Å, respectively. The structure of DgkA was a proof-of principle study with a detailed comparison of samples obtained either after P-CF expression or after inclusion body production in E. coli cells. A comparable X-ray diffraction was ﬁnally obtained with both samples[18].

NMR structural approaches proﬁt most from the labeling oppor-tunities enabled by CF expression. Solution NMR spectra of CF syn-thesized membrane proteins were obtained in different hydrophobic environments such as detergents[52,115], amphipols

[116], nanodiscs[76]or bicelles[117]. The NMR structure of prote-orhodopsin was solved with D-CF synthesized protein in presence of optimized detergent mixtures[16]. Structures of a subunit of the Alzheimer’s disease related protease presenilin-1, of membrane integrated domains of various signal kinases and of several small human integral membrane proteins were obtained with P-CF syn-thesized samples [60,62,63]. Future drug screening approaches based on combinations of NMR analysis of P-CF expressed mem-brane proteins and molecular simulation could thus become feasi-ble[118,119].

Structural dynamics is often crucial for protein–protein or pro-tein–ligand interactions as well as for enzymatic catalysis or trans-port cycles. Spectroscopic methods like NMR and EPR can provide closer insights into these processes. Both techniques require label-ing with spin probes or isotopes and pulsed EPR experiments like PELDOR became the method of choice for the determination of structural dynamics. Several dynamic features of membrane pro-teins have been investigated by these methods in the last years

[120]. Protocols for labeling are available [120,121] and some examples of in vivo expressed membrane proteins are the analysis of the heterodimeric ABC transporter BmrCD[122]and the vitamin transporter ButB [123]. Spin labeling is often performed with lysine or cysteine speciﬁc coupling chemistry. First data of direct spin label insertion via CF expression were obtained with the incorporation of 57_{Fe into a soluble FeFe hydrogenase} _[124]_.

Protocols developed for the direct insertion of spin labels into CF expressed soluble proteins could open the door for similar future studies with membrane proteins[125].

Structural characterization by advanced single particle electron microscopy techniques will come into focus in the near future

[126]. Well-resolved structures of larger assemblies such as the CF synthesized 540kDa ATP–synthase complex of C. thermarum can be obtained[15]. In addition, much lower amounts of samples

are required for electron microscopy and whole virus particles syn-thesized in even mammalian lysates have been visualized [127]. The continuously increasing resolution of electron microscopy could allow to analyze even smaller particles such as complexes of membrane proteins in nanodiscs. Protein inserted into nan-odiscs can be analyzed by atomic force microscopy as well as force spectroscopy, revealing their native conformation [39,94]. The evolving ﬁeld of native mass spectrometry of membrane proteins and membrane protein complexes[128]especially in combination with the nanodisc technology[129,130]gives a further promising perspective for the characterization of CF expressed membrane proteins.

8. Outlook and perspectives

E. coli lysates have already become the workhorse in the field of CF systems and the versatility of applications will continuously grow. Evaluation of lysate blends combining benefits of different organisms is among the future developments and it will further boost the production of difficult biomolecules. Based on valuable intrinsic features such as the cost-effective preparation of lysates, the robustness and reliability of the translation machinery and the immense accumulated knowledge in recombinant protein expression, CF systems with E. coli lysates will further strengthen their role as standard platform for preparative scale protein pro-duction. Industrial scale applications such as the production of antibody-drug conjugates and the design of hybrid biosynthetic pathways with new and advanced properties in CF environments are already emerging. CF expression completely re-designed mem-brane protein production and the elimination of many restrictions caused by the need to maintain cell viability opened numerous new avenues of applications. Multitudes of chemically synthesized compounds can now directly be used for the co-translational stabi-lization of membrane proteins. The systematic screening of lipid effects on membrane proteins by the combination of nanodiscs with CF reactions is just one example for the excellent synergies in this new field of synthetic biology.

Acknowledgements

This study was funded by the Collaborative Research Center (SFB) 807 of the German Research Foundation (DFG). Support was further provided by Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI) and funded by national member subscriptions.

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