Cell-free expression profiling of E. coli inner membrane proteins

RESEARCH ARTICLE

Cell-free expression profiling of E. coli inner membrane

proteins

Daniel Schwarz1�, Daniel Daley2, Tobias Beckhaus3, Volker Dotsch1 and Frank Bernhard1

1 Center for Biomolecular Magnetic Resonance, Goethe-University of Frankfurt/Main, Institute for BiophysicalChemistry, Frankfurt/Main, Germany

2 Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University,Stockholm, Sweden

3 Goethe-University of Frankfurt/Main, Institute of Pharmaceutical Chemistry, Frankfurt/Main, Germany

Received: July 7, 2009

Revised: January 27, 2010

Accepted: February 1, 2010

The high versatility and open nature of cell-free expression systems offers unique options to

modify expression environments. In particular for membrane proteins, the choice of co-

translational versus post-translational solubilization approaches could significantly modulate

expression efficiencies and even sample qualities. The production of a selection of 134

a-helical integral membrane proteins of the Escherichia coli inner membrane proteome

focussing on larger transporters has therefore been evaluated by a set of individual cell-free

expression reactions. The production profiles of the targets in different cell-free expression

modes were analyzed independently by three screening strategies. Translational green

fluorescent protein fusions were analyzed as monitor for the formation of proteomicelles

after cell-free expression of membrane proteins in the presence of detergents. In addition, two

different reaction configurations were implemented and performed either by robotic semi-

throughput approaches or by individually designed strategies. The expression profiles were

specified for the particular cell-free modes and overall, the production of 87% of the target list

could be verified and approximately 50% could already be synthesized in preparative scales.

The expression of several selected targets was up-scaled to milliliter volumes and milligram

amounts of production. As an example, the flavocytochrome YedZ was purified and its

sample quality was demonstrated.

Keywords:

Detergent solubilization / Flavocytochrome / Green fluorescent protein / Inner

membrane proteome / Technology / Transport proteins

1 Introduction

Membrane proteins (MPs) correspond to 20–40% of the

open reading frames in average genomes, but they still

account for less than 1% of the known high-resolution

protein structures [1]. Their typical organization into

multiple hydrophobic transmembrane segments (TMSs),

toxicity and stability problems in cellular environments,

Correspondence: Dr. Frank Bernhard, Center for Biomolecular

Magnetic Resonance, Goethe-University of Frankfurt/Main,

Institute for Biophysical Chemistry, Max-von-Laue-Str. 9,

D-60438 Frankfurt/Main, Germany

E-mail: [email protected]

Fax: 149-69-798-29632

Abbreviations: CD, circular dichroism; CECF, continuous

exchange cell-free; CF, cell-free; CMC, critical micellar concen-

tration; D-CF, cell-free expression in the presence of detergents;

DDM, n-dodecyl–b-D-maltoside; DPC, dodecyl-phosphocholine;

FMN, riboflavin 50-monophosphate; GFP, green fluorescent

protein; LMPC, 1-myristoyl-2-hydroxy-sn-glycero-3-phos-

phocholine; LMPG, 1-myristoyl-2-hydroxy-sn-glycero-3-[phos-

pho-rac-(1-glycerol)]; LPPG, 1-palmitoyl-2-hydroxy-sn-glycero-3-

[phospho-rac-(1-glycerol)]; MP, membrane protein; P-CF, cell-

free expression as precipitate; RM, reaction mixture; SB3-14, 3-

(N,N-dimethylmyristylammonio)propanesulfonate; TMS, trans-

membrane segment

�Current address: Department of Molecular Interactions and Biophy-

sics, Merck KGaA, Dormstadt, Germany

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

1762 Proteomics 2010, 10, 1762–1779DOI 10.1002/pmic.200900485

inefficient insertion into membranes as well as harsh

extraction procedures make overexpression and purification

attempts of MPs still far more challenging if compared with

soluble proteins [2]. Cell-free (CF) expression techniques

have emerged in recent times as new tools for the rapid

production of MPs in a variety of different conditions [3].

The elimination of many intrinsic expression problems by

using CF environments provides a general basis for high

success rates of MP production.

The structural folding of MPs is stabilized by hydro-

phobic compounds and the selected environments can

therefore play pivotal roles in sample quality. The open

accessible nature of CF systems allows the supplementation

of surfactants, detergents or lipids even in high concentra-

tions [4]. Depending on the provided environment, several

modes for the CF production of MPs can thus be approa-

ched. Standard CF systems are almost completely devoid of

membranes and synthesized MPs will therefore instantly

precipitate, described as the mode of cell-free expression as

precipitate (P-CF). It should be noted that MPs produced in

this P-CF expression mode must not necessarily form

completely unfolded aggregates. In contrast, even complex

polytopic MPs could be reconstituted into functional

proteins after P-CF mode expression [5, 6]. A unique option

provided by CF systems is the expression of MPs in the

presence of supplied detergents, described as the mode

of cell-free expression in the presence of detergents (D-CF)

[7, 8]. Freshly translated MPs have the possibility to fold and

to stay soluble by insertion into micelles. Proteomicelles are

required for crystallization and NMR studies, thus making

the D-CF expression of MPs highly interesting for structural

approaches. Alternatively, the insertion of CF synthesized

MPs into supplied liposomes appears to be feasible and

efficient for an increasing number of targets [9, 10]. This

cell-free expression in the presence of lipids expression

mode mostly resembles the natural environment of MPs

and will become important for their functional character-

ization. However, specific protocols still have to be devel-

oped for the efficient insertion (translocation) of individual

MPs.

The increasing variety of CF expression tools for MP

production generates many options in the design of

expression strategies. The selected CF mode as well as the

reaction set-up in either batch configuration or in the more

complicated but higher efficient continuous exchange cell-

free (CECF) configuration are important parameters [11, 12].

In addition, CF reaction protocols are continuously subject

of modifications as numerous potentially beneficial addi-

tives can be supplemented in multiple combinations and at

any time point of the reaction. Results obtained after CF

expression with different modes, configurations and proto-

cols are therefore difficult to compare and often depend on

the individual characteristics of the expressed MP. The

intention of this study was therefore to evaluate CF

expression success rates and efficiencies of a larger

and representative variety of MPs in systems based on

Escherichia coli extracts. A particular emphasis was put on

the differentiation between the P-CF and the D-CF modes of

expression and on addressing some basic questions: (i) Are

general limitations of CF expression of MPs obvious,

defined by size, function, topology or number of TMSs? (ii)

Are success rates and MP production efficiencies different

in the P-CF and D-CF modes? (iii) Can prime parameters be

identified that define the quality of individual MPs?

Bioinformatic analysis of the E. coli proteome indicates

that approximately 1000 of the 4288 predicted genes encode

integral inner MPs [13]. For a number of 737 prevalent

a-helical topology containing at least two TMSs was identi-

fied [13]. A representative subset of 134 targets covering

diverse MP families, protein sizes of up to 1000 amino acids

and of up to 15 predicted TMSs was selected for an

expression analysis. At first, reactions were performed in

batch configuration in the D-CF mode (batch-D-CF) by

using a robotic device. This analytical scale throughput

approach analyzed the general express-ability of the targets.

In parallel, the more productive CECF configuration in

combination with the D-CF mode (CE-D-CF) as well as with

the P-CF mode (CE-P-CF) of expression was employed. The

production yields of the targets were compared and their

solubilization efficiencies in detergent were evaluated.

Finally, the sample quality of a cofactor containing MP was

evaluated after expression in different conditions. The

documented efficiencies and characteristics of the different

CF expression modes and configurations provide guidelines

for further proteomic scale CF expression approaches and

for subsequent characterizations of the analyzed targets.

2 Materials and methods

2.1 DNA techniques

The E.coli MP-green fluorescent protein (GFP) library was

constructed as described previously [14] and the MPs were

expressed from the vector pET28(a1) containing a C-term-

inal TEV protease recognition sequence (ENLYFQ/G)

followed by a C-terminally His8-tagged GFP.

The coding sequences for the E. coli AmtB, LacY, MdtB,

MelB, NhaA and PutP were cloned with BamHI and XhoI(NewEnglandBiolabs, Frankfurt, Germany) in a derivative of

pET21a(1) (Merck Biosciences, Darmstadt, Germany) and

expressed with an N-terminal T7-tag and a C-terminal

poly(His)10-tag. The coding sequence of YedZ was NdeI/

XhoI (NewEnglandBiolabs) cloned into a derivative of

pIVEX2.3MCS (Roche Diagnostics, Penzberg, Germany)

encoding for a C-terminal poly(His)10-tag.

2.2 CF expression

The preparation of T7-RNA polymerase, S-30 extract, plas-

mid DNA and the final set-up of analytical as well as

Proteomics 2010, 10, 1762–1779 1763


preparative scale CECF reactions were performed as

described in detail [15]. The final Mg21 and K1 concentra-

tions were optimized for each new batch of S30 extract. For

CF batch reactions, they have been in the ranges of

6–15 mM Mg21 and 250–300 mM K1, respectively. For

CECF reactions, Mg21 optima have been in between 13 and

15 mM and K1 optima in between 280 and 300 mM. For

toxicity analysis, expression negative MP-GFP templates

(0.015 mg/mL) were mixed with GFP template (0.03 mg/

mL) in a mass ratio of 2:1.

Batch reactions were performed in 96 well V-shape MTPs

(PS-microplate 96 well V-shape ON 651101, Greiner Bio-

One, Frickenhausen, Germany) in a final reaction volume of

25mL and a temperature of 321C at a shaking frequency of

9 Hz (TECAN-shaked plate incubator). The standard reac-

tion mixture (RM) contained the following components:

1.2 mM ATP, 0.8 mM each of GTP, UTP and CTP, 34mg/mL

folinic acid, 170 mg/mL E. coli tRNA mixture (Roche,

Indianapolis, IN, USA), 15 mg/mL of plasmid vector, 6 U/mL

T7 RNA polymerase, 2 mM each of 20 unlabeled amino

acids, 0.33 mM NAD, 0.26 mM CoA, 300 mM potassium

glutamate, 10 mM ammonium glutamate, 10 mM magne-

sium glutamate, 1.5 mM spermidine, 1 mM putrescine,

4 mM sodium oxalate, and 0.24 volume of S30 extract [16].

Reactions were additionally supplemented with 33 mM

sodium pyruvate, 1 mM putrescine, 1.3 mM ATP, 0.9 mM

each of GTP, UTP and CTP, 0.2 mM NAD, 10 U/mL T7 RNA

polymerase, 30 mM PEP and 1 mM DTT in order to enhance

expression yields.

Batch reactions were pipetted on a TECAN Freedom EVO

200/8 device equipped with an eight channel liquid hand-

ling (4mL� 1000 mL and 4 mL� 50 mL syringes) and two

transport arms (Tecan, M.annedorf/Z .urich, Switzerland).

Stock solutions were kept on cooling carriers upon pipet-

ting. Each experiment was initially performed in duplicates.

If results were inconsistent with variations 475%, a

second set of two independent experiments was performed.

The shown values represent averages of the two highest

obtained positive results. If a target was not detected at all by

GFP fluorescence or by immunoblotting in at least three

independent sets of expression experiments, it was classified

as not expressed.

2.3 Electrophoresis

For SDS-gel analysis, protein samples supplemented with

SDS sample buffer (300 mM Tris, pH 7.8, 7.5% SDS w/v;

50% glycerol v/v; 25% b-mercaptoethanol v/v; 0.1%

Coomassie Blue w/v), treated for 30 min at 251C and loaded

on 12% or 16.5% w/v Tris/glycine/SDS gels and stained

with Coomassie Blue. For Western blot analysis, the gels

were transferred on a 0.45 mm Immobilon-P PVDF

membrane (Millipore, Eschborn, Germany) in a wet

Western blot apparatus (BioRad, M .unchen, Germany) for

50 min at 350 mA. For Dot blot analysis samples were

directly applied on a nitrocellulose membrane (Sartorius,

Gottingen, Germany) and stored at 371C until the sample

was totally dry. Membranes were blocked for 1 h in blocking-

buffer containing 1�TBS, 4% skim milk powder and 0.05%

w/v Tween 20.

Poly-His-tags were detected with anti-penta His IgG from

mouse (Qiagen, Hilden, Germany) in a 1:2000 dilution.

Membranes were washed three times with 1�TBS and

0.05% Tween 20. As a second antibody anti-mouse IgG

HRP conjugate from goat (Sigma-Aldrich, Taufkirchen,

Germany) was used in a 1:5000 dilution. Immunodetection

of GFP was performed with anti-GFP (26–39) IgG from

rabbit in a 1:2000 dilution followed by goat anti-rabbit IgG

HRP conjugate (Calbiochem Sigma-Aldrich) in a 1:10 000

dilution. Finally, the blots were analyzed by chemilumi-

nescence in a Lumi-imager F1TM (Roche Diagnostics,

Penzberg, Germany).

2.4 Protein purification

Precipitate was removed from the RM after CF expression by

centrifugation at 20 000� g for 10 min. P-CF produced protein

was resolubilized in resolubilization buffer (50 mM Tris (pH

7.8), 150 mM NaCl, suitable detergent) in a final volume equal

to the RM volume. If appropriate, 2 mM DTT was added

freshly. The suspension was incubated for 2 h at room

temperature or at 301C with gentle shaking followed by

centrifugation for 10 min at 20 000� g in order to remove

residual precipitate. In order to reduce detergent or reducing

agent concentrations (e.g. from 1 to 0.1% in case of Brij-78)

and to ensure a better binding to the column in the subse-

quent purification step, resolubilized P-CF and D-CF samples

were diluted 1:10 in IMAC buffer A (A/B/C/D: 50 mM Tris

(pH 7.8), 300 mM NaCl, 20/70/150/300 mM imidazole, 0.03%

n-dodecyl–b-D-maltoside (DDM)/0.1% Brij-58/0.1% Brij-78).

The diluted RM was incubated with 300mL Ni-NTA Superflow

matrix (Qiagen) per 1 mL of RM. Chromatography was

performed with washing steps of 10 column volumes of

IMAC buffer A, B and C. The bound protein was finally eluted

with IMAC buffer D in steps of 50% bed volume fraction size.

For sample analysis by SEC, appropriate volumes of the

elution fractions from IMAC were loaded by an AKTApurifier

system (GE Healthcare, M .unchen, Germany) on a Super-

dex200 3.2/30 (flow rate 0.03 mL/min) or Superose6 10/300

(flow rate 0.5 mL/min) column (GE Healthcare) pre-equili-

brated with SEC buffer (50 mM Tris (pH 7.8), 150 mM NaCl,

0.03% DDM/0.1% Brij-58/0.1% Brij-78). If appropriate, 2 mM

DTT was added freshly.

Purification of the apo form of YedZ followed the general

procedure as described above. To obtain cofactor bound

YedZ (holo), hemin and/or riboflavin 50-monophosphate

(FMN) were added co-translational (CF-reaction) and/or

post-translational to the resolubilization- and/or IMAC-

buffer A in a final concentration of 100 mM. Efficiency of

holo-form YedZ refolding was evaluated by determination of

1764 D. Schwarz et al. Proteomics 2010, 10, 1762–1779


protein to cofactor ratio (hemin; A280/A445) using SEC

profiles (co-elution) and UV/vis spectra under reducing

conditions.

2.5 Spectroscopical methods

YedZ was purified, dialyzed against circular dichroism (CD)-

buffer and oxidized in 2 mM [Fe(CN)6]3�. Subsequently,

absorption spectra were measured with a light spectro-

photometer (UV-550 Jasco spectrophotometer (Jasco Labor-

technik, Gross-Umstadt, Germany)), before and after

reduction with sodium dithionite (final concentration

approximately 10 mM).

CD spectroscopy was performed with a Jasco J-810

spectropolarimeter (Jasco Labortechnik) in 10 mM sodium

phosphate, pH 8.0, 0.5 mM DTT, and with the appropriate

detergent. Assays were carried out at standard sensitivity

with a bandwidth of 2 nm and a response of 4 s. The data

pitch was 0.2 nm and the scanning rate 100 nm/min. The

spectra were recorded from 190 to 260 nm at 201C in a

cuvette of 1 mm cell length. The presented data are the

average of five scans. The a-helical content of proteins was

calculated according to [17].

Folded GFP (red shifted mutant) concentrations were

determined using fluorescence spectroscopy [18]. 297mL

of assay buffer (20 mM Tris, pH 7.8; 150 mM NaCl)

were mixed with 3mL for 1 h on ice-incubated sample

and aliquoted in a 96-well plate (96F Nunclon Delta Black

Microwell SI, ON 137101 (Nunc, Langenselbold, Germany)).

Method parameters as defined in TECAN Magellan 5.03:

excitation wavelength: 485 nm; emission wavelength:

510 nm; number of reads: 10; unit: RFU. RFU 5 (3.4197

� (GFPconc(mg/mL)))197.551 (valid signal range:

160–1000 RFU).

2.6 MS

Samples for MALDI TOF and MALDI TOF/TOF MS were

obtained from Coomassie-stained protein bands excised

from gels after SDS-PAGE. Samples were destained,

reduced and alkylated as described using a Microlabs Star

digestion robot (Hamilton, Bonaduz, Switzerland) for

sample processing [19]. The supernatant was collected after

overnight trypsin digestion. The remaining peptides were

extracted with 50% v/v ACN/0.3% TFA and two times with

100% ACN. All fractions were pooled, dried in a vacuum

centrifuge and stored at �201C prior to MS analysis. Dried

samples were dissolved in 4 mL 70% v/v ACN, 0.5% v/v TFA.

Aliquots of 0.5 mL sample and 0.5 mL matrix (3 mg/mL

CHCA (Bruker, Bremen, Germany) in 70% v/v ACN,

0.5% v/v TFA) were spotted consecutively on a stainless steel

MALDI target (Applied Biosystems (ABI), Darmstadt,

Germany) and dried under ambient conditions. MS experi-

ments were performed on a 4800 MALDI TOF/TOFTM

Analyzer (Applied Biosystems). The acquisition range was

set to 900–3500 Da. In total, 1500 scans were accumulated

for each PMF. A standard peptide calibration mix (4700

Mass Standards Kit, Applied Biosystems, MDS SCIEX) was

used for external calibration. Selected peaks of ManZ were

fragmented to verify PMF identifications.

All MS spectra were smoothed, noise-filtered and mono-

isotopically labeled using 4000 Series ExplorerTM software

(Applied Biosystems). Mono-isotopic peaks with a S/N >5

were annotated using peaks to MASCOT. The generated

peak lists were searched against a custom database

containing 26 constructs using MASCOT search engine

version 2.2 (Matrix Science, UK). Searches were done with

tryptic specificity allowing one missed cleavage. Mass

tolerance was set to 60 ppm. Carbamidomethylation of

cysteine was set as fixed and oxidation of methionine as

variable modifications. MS/MS spectra were processed

and searched similarly except that the S/N threshold for

the mono-isotopic labelling was six and that the maximal

number of allowed peaks was 50. Searches were done

using MALDI TOF/TOF as instrument type and a 60 ppm

mass tolerance for the precursor and 0.5 Da for the frag-

ments. Database, search engine and search parameters

were used as for the PMF search. Peptides were considered

as identified when the scoring value exceeded the identity-

or extensive homology threshold value calculated by

MASCOT or the significant homology value. Spectra with

scores below the significant homology value were inspected

manually.

3 Results

3.1 Target selection from the E. coli inner

membrane proteome

A number of 134 MP targets were selected in order to obtain

a representative distribution according to molecular mass,

number of predicted TMSs and function (Fig. 1). A parti-

cular concern was on the evaluation of larger MPs involved

in transport processes. The important class of influx and

efflux transporters was therefore overrepresented with

together 59% if compared with 40% in the E. coli inner

membrane proteome. The heterogeneous class of func-

tionally not assigned MPs was underrepresented with only

19% (39% in E. coli). Targets involved in metabolism and

biogenesis accounted to 10 and 7%, which is in correspon-

dence to their prevalence in the E. coli proteome. MPs of

further miscellaneous functions account for 5% of the

targets. The proposed cytoplasmic localization of the protein

termini of the selected library was 54% for the N-termini

(Nin) and 62% for the C-termini (Cin) as previously deter-

mined with corresponding GFP and PhoA fusions [13]

(Fig. 1). More than 60% of the selected targets are composed

of 4300 amino acids and more than 75% contained 44

proposed TMSs.

Proteomics 2010, 10, 1762–1779 1765


3.2 GFP as expression monitor for MPs in CF

systems

A C-terminal GFP tag was used as monitor for the

quantification of CF MP expression in the D-CF mode

and increased the molecular mass of each analyzed MP

for approximately 27 kDa. GFP is known as suitable

expression monitor for MPs in vivo, however its use in

CF expression has not been studied yet [18, 20]. The

folding of nascent GFP appeared to be sensitive to the

presence of certain detergents in the CF reaction. GFP

fluorescence was almost completely abolished in the

presence of 0.2% decyl-maltoside, 0.1% DDM or 0.4%

digitonin, while the efficiencies of GFP expression was

similar in all experiments as analyzed by SDS-PAGE.

Quenching effects could be excluded as addition of the

detergents to already folded and purified GFP did not affect

its fluorescence. The detergents Brij-58 and Brij-78 reduced

GFP fluorescence only down to some 30%, while this

reduced GFP fluorescence remained constant upon further

increased detergent concentrations from 0.2 to 1.2%. A Brij-

78 concentration of 0.6% was selected for further monitor-

ing MP-GFP expression in the D-CF mode. However,

considering the observed reduced folding rate of GFP, only

approximately one-third of the synthesized MP-GFP

proteins can be detected. Furthermore, the fluorescence

background in our D-CF system was fixed to an amount

corresponding to 20 mg/mL GFP.

3.3 CF expression analysis of MPs with an

automated throughput system

All templates were expressed without N-terminal tags and

with C-terminal fusions of GFP followed by a poly(His)8-tag

[13, 14]. Three complete and independent expression

screens were performed (Fig. 2). First, the expression of the

library was determined in batch-D-CF reactions and

analyzed by GFP fluorescence. This initial screening was

performed in V-shaped standard 96-well microplates with

reaction volumes of 25mL (Fig. 2). The batch reaction

protocol was based on the published ‘‘Cytomim’’ system

with some modifications [16]. PEP was added to 30 mM final

concentration and NTP concentrations were increased for

better efficiencies. The expression of all MP targets was

screened with the protocol given in Section 2. First, a

mastermix containing all constant compounds of the

screening reactions except NTPs was made. Pipetting of the

mastermix for one 96-well plate by an integrated eight-

channel liquid handling robotic arm took approximately

20 min. Screening compounds like DNA templates were

dispersed first to each well of the 96-well plate, followed by

the prepared mastermix. NTPs were finally added in order to

start the reactions.

In batch-D-CF reactions in the presence of 0.6% Brij-78,

the expression of a total of 84 MPs representing 63% of all

targets could be detected by GFP fluorescence (Table 1). The

calculated production levels based on GFP fluorescence

Figure 1. MP target selection for the CF expression screen. (A) Targets classified according to the proposed function. (B) Classification

according to physical parameters.

Figure 2. Screening scheme of

the CF expression profiling.



Table 1. CF production of E. coli inner MPs

Protein aaa) TMSb) Function D-CFc) batch (nM)d) D-CFe) CECF (nM)d) P-CFf) ECg)

AbgT 508 13 T/aminobenzoyl-glutamate transport 0 0 0 0AgaC 267 7 T/N-acetylgalactosamine permease IIC-1 2 (775) 1 (249) 2 2iAmpE 284 4 S/b-lactamase regulation 2 (559) 2 (923) 2 1oAmtB 428 11 T/ammonium uptake 1 (256) 1 (330) 3 1iAraJ 394 12 Unknown 0 0 0 2iAroP 457 12 T/aromatic amino acid transport 1 (119) 1 (74) 2 2iAtoE 440 10 T/short-chain fatty acid transport 2 (434) 3 (4882) 3 2oAtpI 247 4 M/translocation of protons 1 (4) 2 (1026) 3 2iBcr 396 12 E/bicyclomycin resistance 0 1 (4) 1 2iBrnQ 439 12 T/branched-chain amino acid transport 1 (159) 2 (489) 3 2iChbC 452 10 T/N,N0-diacetylchitobiose transport 3 (1715) 2 (785) 2 3iCmr 410 12 E/multidrug translocase 0 1 (82) 0 3iCodB 419 12 T/cytosine permease 0 1 (78) 2 3iCrcB 127 4 unknown 1 (289) 3 (3889) 3 2oCreC 474 3 S/sensor protein 0 0 2 2iCyoE 296 7 B/protoheme IX - heme conversion 0 1 (185) 3 1oDcuA 433 10 T/C4-dicarboxylate transport 1 (19) 2 (397) 2 1oDcuB 446 10 T/C4-dicarboxylate transport 0 1 (304) 1 2oDmsC 287 8 M/dimethyl sulfoxide reductase chain C 0 1 (52) 0 2oDsdX 445 12 T/permease 2 (578) 1 (245) 1 3oEfeU 276 7 5 YcdN, T/ferrous iron uptake 0 2 (863) 3 0EmrE 110 4 E/multidrug transporter 3 (2249) 3 (8663) 3 3oExuT 472 12 T/hexuronate transporter 0 1 (23) 2 3iFdoI 211 4 M/cytochrome b556 subunit 1 (289) 3 (5530) 2 2iFepD 334 9 T/ferric enterobactin transport 0 1 (45) 0 2iFliF 552 2 F/flagellar M-ring protein 0 0 0 0FrdD 119 3 M/fumarate reductase subunit 3 (1530) 2 (2863) 2 3oFrvB 483 10 T/sugar phosphotransferase subunit 0 1 (4) 1 0GlpT 452 12 T/glycerol-3-phosphate uptake 1 (137) 1 (52) 1 2iGltK 224 6 T/aspartate/glutamate transporter subunit 1 (89) 3 (7630) 3 2iGltS 401 12 T/sodium-dependent glutamate uptake 0 1 (111) 1 1oGntU 446 12 T/gluconate utilization system subunit 0 1 (100) 1 2oGspO 225 7 B/type 4 prepilin peptidase 1 (136) 3 (14 386) 3 1oGudP 450 12 5 YgcZ, T/D-glucarate uptake 2 (418) 1 (186) 1 3iHdeD 190 6 Unknown 2 (564) 1 (339) 2 2iHisQ 228 5 T/histidine transport 2 (664) 3 (7600) 3 0HybB 392 10 M/putative b-type cytochrome 1 (22) 1 (30) 1 1iHycD 307 8 M/formate hydrogenlyase subunit 4 2 (516) 1 (189) 1 2oHyfE 216 7 M/hydrogenase-4 component E 1 (68) 3 (5293) 3 2iKdgT 327 10 T/2-keto-3-deoxygluconate permease 1 (6) 0 3 1iLacY 417 12 T/b-galactoside transport 1 (54) 1 (114) 2 2iLgt 291 5 B/prolipoprotein diacylglyceryl transferase 1 (39) 1 (204) 3 3iLldP 551 12 5 YghK, T/L-lactate transporter 2 (561) 1 (32) 1 2oMacB 648 4 5 YbjZ, E/macrolide export subunit 2 (489) 1 (182) 1 0ManZ 286 1 T/mannose transport 1 (75) 2 (932) 3 1oMarC 221 6 E/multiple antibiotic resistance 0 1 (182) 3 1oMdtL 391 12 5 YidY, E/chloramphenicol resistance 1 (164) 1 (4) 3 2iMelB 469 12 T/melibiose transport 1 (104) 2 (454) 2 2iMglC 336 8 T/galactoside transport subunit 1 (32) 1 (125) 0 2iMhpT 403 12 T/3-phenylpropionate transporter 0 2 (1071) 1 0MntH 412 11 5 YfeP, T/H1-stimulated Mn21 uptake 1 (158) 2 (2047) 3 1oMreD 162 5 B/rod shape formation of cells 1 (139) 1 (50) 0 1iMtr 414 11 T/tryptophan transport 1 (68) 2 (932) 1 2oNanT 496 12 T/sialic acid transport 1 (171) 2 (1786) 3 2iNarI 225 5 M/nitrate reductase subunit 1 (15) 2 (2068) 1 1iNhaA 388 10 T/Na1/H1 antiporter 0 1 (5) 3 1iNhaB 513 12 T/Na1/H1 antiporter 0 0 0 0NorM 456 12 E/multidrug efflux pump 0 0 2 2iNuoK 100 2 M/NADH-quinone oxidoreductase subunit 1 (171) 1 (65) 1 1i-NuoL 613 14 M/NADH-quinone oxidoreductase subunit 0 0 2 2o

Proteomics 2010, 10, 1762–1779 1767


Table 1. Continued


NupG 418 12 T/nucleoside transport 0 0 0 1iPnuC 239 6 T/NMN transport 3 (1,471) 3 (14 454) 2 2iPotE 439 12 T/putrescine-ornithine antiporter 1 (82) 2 (393) 3 2iProY 457 12 T/proline-specific permease 0 0 1 2iPutP 502 12 T/sodium-dependent L-proline uptake 1 (54) 2 (864) 3 2iRarD 296 10 T 1 (285) 1 (43) 2 3iRcsC 949 2 S/sensor kinase 0 0 2 0RhaT 344 10 T/uptake of L-rhamnose 1 (75) 1 (93) 1 2oSapC 296 6 T/peptide transport 0 0 0 2iSdaC 429 11 T/serine import 1 (29) 2 (379) 3 2oSecG 110 2 B/protein export 0 1 (61) 0 2oSetC 394 12 5 YicK, E/sugar efflux 0 0 0 1iSugE 105 4 E/quaternary ammonium compound efflux 3 (2564) 3 (27 015) 3 3oTehA 330 8 E/potassium tellurite resistance 1 (4) 2 (2 750) 3 1iThiP 536 12 T/thiamine import subunit 0 0 0 0TnaB 415 11 T/tryptophan transport 1 (18) 1 (186) 3 1oUbiA 290 8 B/3-octaprenyl-4-OH-benzoate synthesis 0 2 (357) 3 2oUbiB 546 2 5 AarF, B/ubiquinone biosynthesis 0 0 2 0UgpA 295 6 T/sn-glycerol-3-phosphate transport 0 0 0 2iUidB 457 11 T/glucuronide permease 1 (168) 1 (39) 1 2iWzc 720 2 L/colanic acid synthesis 1 (157) 1 (68) 3 1iXylH 393 11 T/D-xylose transport 0 0 0 2iYaaH 188 6 Unknown 2 (593) 3 (5097) 3 1iYabM 392 12 E/sugar efflux activity 1 (189) 1 (96) 3 2iYadS 207 7 Unknown 0 0 0 1iYbaN 125 4 Unknown 2 (329) 2 (3204) 3 2iYbbJ 152 3 Unknown 2 (471) 2 (3232) 2 2iYbhI 477 11 T 1 (143) 1 (71) 1 0YbhQ 136 4 Unknown 1 (136) 2 (725) 1 2iYbiF 295 10 T 0 0 0 3iYbiP 527 4 Unknown 2 (518) 2 (843) 3 2oYbjE 299 6 Unknown 0 0 0 0YbjL 561 10 T 0 1 (14) 0 0YchQ 130 4 5 SirB2, unknown 2 (579) 3 (18 661) 1 1oYciS 102 2 Unknown 1 (249) 2 (482) 1 2iYdcZ 149 5 Unknown 1 (50) 1 (125) 3 1iYdeD 306 10 E/amino acid metabolite efflux pump 0 0 0 3iYdgF 121 4 E/SMR YdgEF complex subunit 1 (186) 3 (4093) 3 1iYdjM 200 2 Unknown 2 (536) 1 (71) 2 3oYeaL 148 4 Unknown 0 0 0YedA 306 10 T 0 0 0 2iYedQ 564 2 B/regulation of cellulose production 1 (58) 1 (82) 0 0YedZ 211 6 M/involved in respiratory chain 3 (2286) 3 (18 761) 2 2iYeeF 452 12 T/amino acid or metabolite transporter 1 (222) 2 (1497) 3 3iYegN 1040 11 E/putative efflux protein 1 (186) 0 3YegT 425 11 T/nucleoside transport 1 (225) 2 (450) 3 2iYehY 385 10 T/ABC-transport component 1 (136) 2 (1422) 2 1iYeiO 393 12 E/sugar efflux transport 1 (161) 0 1 0YfbI 550 12 L/dolichyl-P b-D-mannosyltransferase 1 (4) 1 (136) 1 0YfcC 506 11 Unknown 2 (415) 1 (97) 0 0YfcJ 392 12 Unknown 0 0 0 2iYfiK 195 6 E/cysteine exporter 2 (422) 2 (508) 1 2oYfjW 567 6 Unknown 2 (471) 1 (239) 0 0YgiH 205 5 Unknown 1 (57) 2 (1683) 0 2iYhaH 121 3 Unknown 2 (882) 3 (10 168) 3 2iYhbE 321 10 T 0 1 (22) 0 3iYhbX 541 5 Unknown 2 (572) 1 (15) 1 2oYhfK 700 9 Unknown 0 0 0 0YhiH 911 5 T/putative ABC-transporter 0 1 (4) 3 0YhjK 651 2 S/involved in protein binding events 0 0 0 0



were ranging in between 1 and 60 mg/mL. The soluble

expression of 52 targets was very low in amounts of less

than 300 pmol/mL (o8mg/mL GFP). A further 26 targets

were obtained in medium levels in between 300 and

930 pmol/mL (8–25 mg/mL GFP). Only the six targets ChbC,

FrdD, PnuC, EmrE, SugE and YedZ exceeded calculated

production levels of Z930 pmol/mL (Z25 mg/mL GFP). The

expression of a relatively large number of 50 targets repre-

senting 38% was not detectable, which could at least partly

be due to the low efficiency of the batch configuration in

combination with limited GFP folding.

3.4 Evaluation of MP production in CE-D-CF

reactions

In this screen, targets with expression below the detection

limit in the previous batch-D-CF reactions could additionally

be identified due to the higher efficiencies of the CE-D-CF

reactions. The synthesis of a significant higher number of

101 targets representing 75% and covering almost all posi-

tive targets from the previous batch-D-CF screen were

detected (Table 1). The additional 17 MPs were all rated in

lower production levels, indicating that indeed detection was

probably not sensitive enough for these targets in the batch-

D-CF screen. It should be noted that for some 30% of the

targets detected in the CE-D-CF screen, the expression levels

of soluble MP-GFP protein were not higher if compared

with the corresponding batch-D-CF reactions. Productivity

of the CF reaction was therefore not the limiting factor in

those cases.

With the 16 targets AtoE, CrcB, EmrE, FdoI, GltK, GspO,

HisQ, HyfE, PnuC, SugE, YaaH, YchQ, YdgF, YedZ, YhaH

and YicG yields of Z3.7 nmol/mL (Z100 mg/mL GFP) of

soluble MP-GFP protein were achieved (Table 1). While

PnuC, EmrE, SugE and YedZ also belong to the best

soluble expressing targets from the batch-D-CF screen,

for all the others a significant increase in the production

levels in the CE-D-CF reaction can be noted. The vast

majority of this group has less than 300 amino acids

and is almost completely inserted into membranes with only

small predicted loop regions (Fig. 3). A single exception is

the transporter AtoE having a higher molecular mass of

48 kDa and with ten predicted TMSs also obviously some

larger loops. The second group of 32 MPs with expression

levels in between 0.35 and 3.7 nmol/mL (10–100 mg/mL

GFP) is much less homogenous and covers an equal

number of 14 small as well as of 14 mid-sized MPs, but also

four targets exceeding 500 amino acids (Fig. 3). Some MPs

of this group like AmpE, ManZ or YbiP contain only rela-

tively few predicted TMSs which indicates that the presence

of larger loops principally does not prevent solubilization in

the D-CF mode. For the largest group representing almost

40% of the library only spurious GFP folding was detected

and a strong bias toward larger targets can be noted. Also

the group of the 33 not detected targets is clearly dominated

by larger MPs and only ten targets consist of 300 amino

acids.

Table 1. Continued


YicG 205 7 Unkown 2 (303) 3 (4036) 1 1iYicL 307 10 T 0 0 0 2iYidE 553 10 T 2 (611) 2 (383) 0 0YidK 571 15 T/sodium:solute symporter 1 (68) 2 (607) 2 2iYijD 119 4 Unknown 0 2 (3107) 3 0YijE 301 10 T 1 (129) 1 (12) 2 2iYjcC 528 2 Unknown 2 (529) 1 (12) 1 0YjcE 549 12 T/Na1/H1 exchanger 2 (547) 1 (75) 0 0YjdB 541 5 M/involved in cell division 2 (643) 1 (179) 3 0YjjL 453 10 T/phthalate permease family 1 (72) 0 2 1iYqfA 219 7 M/involved in hemolysis 0 0 0 2oYrdE 129 4 Unknown 1 (197) 1 (44) 1 0YtfF 324 10 Unknown 0 0 0 3iYtfT 341 10 T/ABC-transporter family 0 0 1 2i

a) aa, Number of amino acids.b) Number of predicted TMS.c) 0, No folded GFP detected; 1, o300 nM (8 mg/mL); 2, 300 to o930 nM (8 to o25mg/mL); 3, Z930 nM (Z25 mg/mL).d) Numbers represent the average of two independent experiments with a maximum variation of 75%.e) 0, No folded GFP detected; 1, o350 nM (10 mg/mL); 2, 350 to o3700 nM (10 to o100 mg/mL); 3, Z3700 nM (Z100 mg/mL).f) 0, Not detectable on Coomassie Blue stained SDS-PAGE; 1, spurious expression; 2, clearly detectable on Coomassie Blue stained SDS-

PAGE; 3, dominant band on Coomassie Blue-stained SDS-PAGE.g) Expression in E. coli. In (i): putative cytoplasmic C-terminus, quantified with MP-GFP-fusions; Out (ii): putative periplasmic C-terminus,

quantified with MP-PhoA-fusions. GFP and PhoA units were classified as follows. GFP: 0, o2000 units; 1, 2000 to o4000 units; 2, 4000 too10 000 units; 3, 410 000 units. PhoA: 0, o130 units; 1, 130 to o375 units; 2, 375 to o1000 units; 3, 41000.

Proteomics 2010, 10, 1762–1779 1769


3.5 Evaluation of MP production in the P-CF mode

Targets that are poorly or even not at all detectable in the

D-CF mode of expression might still become synthesized

but could fail to solubilize in Brij-78 and the precipitates

remain non-fluorescent. In addition, the expression effi-

ciencies of the targets in the P-CF mode could be different if

compared with the D-CF mode. Only the more efficient

CECF configuration was used and synthesis of the targets in

the CE-P-CF reactions was analyzed by SDS-PAGE

combined with immuno-detection of the GFP tag (Table 1).

In general, the expression of 99 targets representing a total

of 74% could be detected in the CE-P-CF reactions. A

number of 72 targets representing 53% of the complete

library were already detectable as dominant band by

Coomassie Blue staining after SDS-PAGE (Fig. 4) [21].

Preparative scale production efficiencies appear therefore to

be more likely with a higher number of targets in CE-P-CF

reactions. High expression in the D-CF mode almost always

was combined with high expression rates in the P-CF mode.

Two exceptions are YchQ and YicG whose functions are not

assigned yet. Vice versa, quite a larger number of targets of

poor or no detection in the D-CF mode were synthesized at

significant amounts in the P-CF mode (Table 1). In parti-

cular for very large MPs exceeding 500 amino acids such as

RcsC, Wzc, MdtB (YegN), YhiH or YjdB, the CE-P-CF

reaction appears to be the preferred method to produce

preparative amounts of material (Fig. 3). In addition to the

detection of the CF expressed MPs by SDS-PAGE analysis

and immunoblotting, a representative selection of the

targets was identified by PMF after tryptic digests

(Table 2). Peptides covering the N-terminus or origination

close from the N-terminus gave furthermore evidence of

the full-length expression of the corresponding target in

combination with the above-mentioned C-terminal GFP-tag

detection.

The expression of 22 targets could not be detected in all

three screens. If classified by size, six (UgpA,

YadS, YbiF, YbjE, YeaL, YqfA) contain 300 amino acids, 10

(AraJ, NupG, SapC, SetC, XylH, YdeD, YedA, YfcJ, YicL,

YtfF) are in between 301 and 500 amino acids and another

six (AbgT, FliF, NhaB, ThiP, YhfK, YhjK) exceed 500 amino

acids. For three of them (YbiF, YdeD, YtfF), a relatively high

level of expression in E. coli cells was reported, while for the

others, only moderate or no expression in vivo could be

detected (Table 1). One reason for the failure to

detect any expression could be toxic effects of a synthesized

MP to the CF system, e.g. by inactivation of ribosomes. In

order to test this possibility, we co-expressed all 22 negative

targets together with GFP in batch-D-CF (0.6% Brij-58)

reactions by addition of the two corresponding plasmid

templates at a ratio of 1:2. The resulting GFP expressions

were compared with control reactions containing only the

GFP template. With the templates of the three targets AbgT,

FliF and NhaB, a significant reduction of GFP expression to

less than 50% could be observed, indicating a negative

Figure 3. Success of MP-GFP produc-

tion as detected in the P-CF and D-CF

modes of expression. In the D-CF

mode, Brij-58 was used as detergent.

(A) Targets classified after size. (B)

Targets classified after predicted

number of TMS. (C) Targets classified

after proposed position of the

N-terminus. 0, no expression detect-

able; 1, spurious expression; 2, low

level expression; 3, preparative scale

expression.



effect on expression in these cases. With all other targets,

GFP expression was not reduced if compared with the

controls.

3.6 Quality analysis of CF produced large transport

proteins

The majority of the analyzed targets were larger than 300

amino acids and many of them could be successfully

produced at least in the P-CF mode. Obtaining solubilized

protein samples is essential for MP production and thus the

efficiency of detergent solubilization of few representative

larger MPs was analyzed. The 44 kDa ammonia transport

channel AmtB, the 54 kDa proline transporter PutP and the

41 kDa Na1:H1 exchanger NhaA containing 12 predicted

TMSs each as well as the 114 kDa putative multidrug efflux

pump MdtB (YegN) containing 11 predicted TMSs were

selected as models. All targets were expressed without the

large GFP-tag and they only contained a C-terminal poly

(His)10-tag and a small N-terminal T7-tag. Expression

without the C-terminal GFP-tag did not reduce the expres-

sion efficiencies of MP targets but rather resulted in

improved yields in some cases. The individual expression

protocols were further modified for optimal Mg21

(10–20 mM) and K1 (250–350 mM) concentrations and final

yields were 2 mg/mL NhaA, 1.5 mg/mL PutP, 1.5 mg/mL

AmtB and 0.8 mg/mL MdtB.

The re-solubilization of the CE-P-CF produced MPs

was obtained with various detergents including

1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (LMPC),

1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)]

(LMPG), 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-

(1-glycerol)] (LPPG), dodecyl-phosphocholine (DPC) and

3-(N,N-dimethylmyristylammonio)propanesulfonate (SB3-14)

in concentration ranges between 0.125 and 1% (Fig. 5A).

The solubilized MPs were purified by Ni21-chelate chro-

matography and immunoblotting against the terminal

poly(His)10-tag verified the full-length expression of all

targets and evidence of putative dimer (AmtB, NhaA, PutP)

and trimer (AmtB) formations was observed (Fig. 5A). The

soluble expression of the MPs was further analyzed in the

CE-D-CF mode in the presence of different detergents.

Brij-35, Brij-58, Brij-78 and Brij-98 were most effective to

solubilize MdtB and AmtB (Fig. 5B). The non-ionic alkyl-

polyether alcohol Tyloxapol was furthermore quite effective

in the solubilization of the large multidrug transporter

MdtB. Tyloxapol could be supplemented to the CF reaction

in final concentrations of 1% corresponding to 140� critical

micellar concentration (CMC) with no negative effect on the

expression efficiency.

The homogeneity of the MPs PutP, NhaA and MdtB after

expression in different modes was analyzed by analytical

SEC. The MP samples were first purified by Ni21-affinity

chromatography concomitant with a detergent exchange of

the Ni21-NTA immobilized proteins, if appropriate. The

Figure 4. SDS-PAGE analysis of

CF expressed MP-GFP fusions.

Volumes of 2 mL of the resus-

pended insoluble fraction of

CE-P-CF reactions containing

precipitated MP-GFP fusions as

well as co-precipitated impu-

rities were separated on 12%

SDS-polyacrylamide gels and

stained with Coomassie Blue.

Bands representing the indi-

cated synthesized MP-GFP

fusion are marked with arrows.

Additional bands result from

co-precipitated proteins of the

E. coli extract. Many targets do

not separate exactly according

to their calculated molecular

weight due to altered SDS

loading typical for MPs [21]. (A)

Preparative scale expression

levels 2 or 3; (B) production of

only spurious amounts in level

1; (C) Dot blot analysis of

selected targets which have

been invisible by Coomassie

Blue staining. In general, this

group as well as the targets of

level 1 were further subject of

anti-GFP dot blots analysis.

Proteomics 2010, 10, 1762–1779 1771


Table 2. PMF of CF produced MPsa)

Protein [#aa] Position Mass (M1H) Error (ppm) Amino acid sequenceb) Mc)

AmtB [428] 1–12 1254.5752 29 –.MASMTGGQQMGR.G1–12 1270.5387 4 –.MASMTGGQQMGR.G M11–12 1286.5553 21 –.MASMTGGQQMGR.G M21–12 1302.5428 15 –.MASMTGGQQMGR.G M32–12 1139.5115 16 M.ASMTGGQQMGR.G M2–12 1155.4979 8 M.ASMTGGQQMGR.G M2

AtoE [440] 219–228 1223.5653 �26 K.LLMEEADFQK.Q219–228 1239.5492 �35 K.LLMEEADFQK.Q M1219–232 1689.8156 �43 K.LLMEEADFQKQLPK.D219–232 1705.7887 �55 K.LLMEEADFQKQLPK.D M1

BrnQ [439] 1–6 785.3857 �29 –.MTHQLRS1–6 801.3994 �5 –.MTHQLRS M11–8 1028.4907 �50 –.MTHQLRSR.D

CyoE [296] 185–200 1801.9547 �37 R.FKDYQAANIPVLPVVK.G251–258 923.4233 �38 R.GYKVADDR.I254–262 1101.5601 �18 K.VADDRIWAR.K

HisQ [228] 44–57 1582.8823 �1 R.LSGLIFEGYTTLIR.G114–134 2195.1323 12 R.GAFMAVPKGHIEAATAFGFTR.G M1122–134 1377.6854 �4 K.GHIEAATAFGFTR.G

Lgt [291] 45–54 1073.5404 �8 R.ANRPGSGWTK.N122–143 2395.2866 �2 K.RSFFQVSDFIAPLIPFGLGAGR.L123–143 2239.2029 5 R.SFFQVSDFIAPLIPFGLGAGR.L144–155 1375.6954 �12 R.LGNFINGELWGR.V156–170 1710.8137 �10 R.VDPNFPFAMLFPGSR.T M1

ManZ [286] 15–22 960.6521 5 K.KLTQSDIR.G16–27 1404.8007 4 K.LTQSDIRGVFLR.S23–39 2057.0479 6 R.GVFLRSNLFQSWNFER.M28–39 1484.7057 9 R.SNLFQSWNFER.M28–54 3193.5444 9 R.SNLFQSWNFERMQALGFCFSMVPAIR.R40–54 1727.8723 17 R.MQALGFCFSMVPAIR.R

NanT [496] 1–12 1497.7222 4 –.MSTTTQNIPWYR.H1–12 1513.6948 �10 –.MSTTTQNIPWYR.H M1

NhaA [388] 1–12 1254.5604 17 –.MASMTGGQQMGR.G1–12 1270.5258 �6 –.MASMTGGQQMGR.G M11–12 1286.5931 50 –.MASMTGGQQMGR.G M21–12 1302.5184 �4 –.MASMTGGQQMGR.G M32–12 1123.5247 24 M.ASMTGGQQMGR.G2–12 1139.4905 �2 M.ASMTGGQQMGR.G M1

PutP [502] 1–12 1254.5713 26 –.MASMTGGQQMGR.G1–12 1270.5502 13 –.MASMTGGQQMGR.G M11–12 1286.5184 �8 –.MASMTGGQQMGR.G M21–12 1302.5176 �5 –.MASMTGGQQMGR.G M32–12 1123.545 42 M.ASMTGGQQMGR.G2–12 1139.4979 4 M.ASMTGGQQMGR.G M12–12 1155.478 �9 M.ASMTGGQQMGR.G M2

SdaC [429] 236–246 1355.5807 �7 K.REEYGDMAEQK.C236–246 1371.5862 1 K.REEYGDMAEQK.C M1316–325 1120.5773 �11 K.SFLGHYLGAR.E326–337 1350.6693 �37 R.EGFNGMVIKSLR.G

UbiA [290] 10–15 756.426 �34 K.LLAFHR.L61–72 1310.5769 �2 R.AAGCVVNDYADR.K81–94 1440.7507 �21 R.TANRPLPSGAVTEK.E81–97 1796.9346 �15 R.TANRPLPSGAVTEKEAR.A

Wzc [720] 5–22 1892.8747 �1 K.VKQHAAPVTGSDEIDIGR.L103–127 2765.3594 �7 K.TVDDLDLDIAVSKNTFPIFGAGWDR.L116–127 1380.7325 46 K.NTFPIFGAGWDR.L138–145 962.5783 38 K.VTTFNRPK.E

YeeF [452] 1–11 1243.6008 13 –.MSHNVTPNTSR.V1–11 1259.5977 14 –.MSHNVTPNTSR.V M11–15 1740.8517 �17 –.MSHNVTPNTSRVELR.K



homogeneity of the samples and the apparent molecular

masses are clearly dependent on the production conditions.

The CF expression modes as well as the selected detergents

used for direct solubilization in the D-CF mode or for

resolubilization in the P-CF mode are prime modulators for

the quality of the MP samples. In addition, detergent

exchange after initial solubilization during affinity purifica-

tion can further trigger sample homogeneity. From the

analyzed conditions, the profile of NhaA samples was best if

the protein was P-CF produced, resolubilized in LMPG and

the detergent subsequently exchanged against Brij-78

(Fig. 5C). The elution profile showed two peaks of

approximately 1.3 MDa and 450 kDa. The determined

molecular mass of empty Brij-78 micelles was with

150–300 kDa relatively large (data not shown). The main

peak at 450 kDa could therefore represent a lower oligomeric

complex of NhaA. Aggregation of PutP could significantly

be reduced if the protein was P-CF produced and solubilized

in DDM. P-CF produced MdtB resolubilized in 0.25%

SDS resulted in completely aggregated protein (Fig. 5C).

Resolubilization of the same sample in 0.25% LPPG or

0.25% LMPC resulted in proteomicelles with molecular

masses of approximately 150 and 200 kDa, considering a

molecular mass of empty LMPG micelles of approximately

50 kDa, this presumably could represent solubilized

MdtB monomers. However, non-symmetrical peak shapes

indicated still not completely homogeneous sample

qualities.

3.7 Sample quality of the CF produced putative

flavocytochrome YedZ

YedZ is a 24 kDa leucine rich integral MP with six putative

TMSs. The protein is reported to bind the cofactors heme bas well as FMN [22]. YedZ is expressed in high amounts in

the P-CF as well as in the D-CF modes and its cofactor

incorporation could easily be monitored by spectroscopic

characterization. The C-terminal GFP-tag was replaced by a

poly(His)10-tag after subcloning into vector pIVEX-2.3d and

the protein was expressed in CE-P-CF reactions. P-CF

produced YedZ can efficiently be resolubilized in LMPG,

LPPG, SDS and DPC. Highly pure apo-YedZ in yields of

approximately 1.2 mg/mL RM could be obtained by solubi-

lization in 1% LPPG, purification on a Ni21-chelate column

and elution in 0.02% DDM (Fig. 6A). SEC of this sample

resulted in symmetrical Gaussian shaped peaks with a

calculated molecular mass of the proteomicelles of 110 kDa

(Fig. 6B). SEC of CE-D-CF (1% Brij-78) expressed YedZ in

0.02% DDM resulted in much broader peaks centred on a

mass of 75 kDa. SEC of the same samples in 0.1% Brij-78

yielded narrow peaks with an estimated mass of 200 kDa

(Fig. 6B). CD spectroscopy of purified YedZ in 0.02% DDM

indicated an a-helical content of 55% and denaturation of

YedZ by heating to 951C resulted in the irreversible

unfolding of the protein with a melting temperature of

�661C (Fig. 6C).

Hemin incorporation was quantified by the ratio of

absorbance at 280/445 nm in the elution peaks after SEC

and UV/vis spectroscopy (Table 3). Holo-YedZ could be

produced co-translationally by addition of hemin and FMN

directly into the RM of CE-D-CF (1% Brij-78) reactions.

Alternatively, hemin and FMN were incorporated post-

translationally by addition of the cofactors to purified apo-

YedZ. Co-translationally produced holo-YedZ was only

stable if the subsequent purification was also performed in

the presence of the cofactors (Table 3). However, the sample

homogeneity as evaluated by SEC appeared not to be satis-

fying. Purification in the absence of cofactors resulted in

loss of hemin and in A280/A445 ratios comparable to the

control protein PutP. Therefore, the post-translational

insertion of the cofactors was approached and incorporation

in combination with satisfying sample quality could be

obtained if CE-P-CF produced apo-YedZ was solubilized in

1% LPPG in the presence of hemin and FMN

and the detergent was subsequently exchanged with DDM

upon binding of the protein to a Ni21 chelate column

(Table 3).

Table 2. Continued

Protein [#aa] Position Mass (M1H) Error (ppm) Amino acid sequenceb) Mc)

1–15 1756.905 17 –.MSHNVTPNTSRVELR.K M175–88 1562.7695 6 R.RYPSAGSAYTYAQK.S76–88 1406.6332 �18 R.YPSAGSAYTYAQK.S

YhaH [121] 7–15 1095.627 �4 K.VLKNYVGFR.G10–15 755.3776 �8 K.NYVGFR.G10–17 968.4747 �32 K.NYVGFRGR.A22–48 3361.6638 �37 K.EYWMFILVNIIFTFVLGLLDKMLGWQR.A M143–48 806.3927 �6 K.MLGWQR.A M1

a) Selected assigned peaks in the mass spectra of selected MPs. Only detected peptides located most proximal to the N-termini of the MPsare shown. Fingerprints were verified from at least two independent samples.

b) Trypsin sites are indicated.c) Methionine oxidation.

Proteomics 2010, 10, 1762–1779 1773


Holo-YedZ formation was verified by cofactor specific

absorption maxima in dependency from its state of oxida-

tion by UV/vis spectroscopy. Oxidizing the coloured holo-

YedZ with ferricyanide resulted in absorption maxima at

412 nm, whereas free unbound hemin has an absorption

maximum at 401 nm. Reduction of holo-YedZ with sodium

dithionite resulted in maxima at 425, 528 and 558 nm

(Fig. 6D). The obtained UV/vis spectra of the CF produced

holo-YedZ exactly correspond to recorded spectra of the

heme b containing YedZ purified after in vivo expression in

E. coli [22].

4 Discussion

Considering a total number of �740 integral a-helical MPs

of the analyzed group in E. coli, our selection represents

some 15% [13]. GFP as a C-terminal fusion-partner of

(membrane-) proteins is an established tool to monitor

expression levels in conventional cellular expression

systems [13, 23, 24]. Correctly folded GFP at the C-termini of

fusion proteins is even speculated to indicate the functional

folding of the N-terminally attached fusion partner [24].

However, it remains to be shown whether a similar corre-

lation can be found upon D-CF expression of MP-GFP

fusions into detergent micelles. GFP represents an excellent

expression reporter in lipidic/cellular/membrane environ-

ments, while its folding appears to be significantly reduced

if synthesized in the presence of detergents. Once folded,

the GFP fluorescence is not affected after supplementation

of detergents (data not shown). Neither the GFP synthesis

rate was affected nor did we observe increased precipitate

formation. The non-fluorescent GFP moiety is therefore

soluble but remained at least partly unfolded. Interestingly,

this negative effect of detergents on GFP folding does widely

not depend on the added detergent concentrations exceed-

ing CMC. As only the micelle concentration increases while

the concentration of detergent not forming micelles stays

constant, this indicates that interaction of nascent GFP with

single detergent molecules and not with micelles might

inhibit complete folding. However, using specific Brij deri-

vatives still allows the detection of approximately one third

Figure 5. Solubilization of CF expressed large transporters. Volumes of 1.5 mL of the RM were separated on 12% SDS-Tris-glycine gels and

immunoblotted with anti-(His)5 antibodies. (A) Resolubilization of CE-P-CF expressed MdtB and NhaA and PutP. (B) CE-D-CF expression of

MdtB and AmtB. Supplied detergents and their final concentrations w/v in the D-CF reaction are indicated. S, supernatant; p, pellet; B35,

Brij-35; B58, Brij-58; B78, Brij-78; B98, Brij-98; DDM, n-dodecyl–b-D-maltoside; DPC, dodecyl-phosphocholine; LMPC, 1-myristoyl-2-

hydroxy-sn-glycero-3-phosphocholine; LMPG, 1-myristoyl-2-hydroxy-sn-glycerol-3-[phosphor-rac-(1-glycerol)]; LPPG, 1-palmitoyl-2-

hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)]; TXP, Tyloxapol; TX100, Triton X-100; SB3-14, 3-(N,N-dimethylmyr-

istylammonio)propanesulfonate. (C) Elution profiles of NhaA (Superose 6 10/300), PutP and MdtB (Superdex 200 3.2/300) after SEC. V0

indicates the void volume.



of the synthesized MPs, and this sensitivity was sufficient to

already identify the expression of 62% of our targets in the

initial robotic batch-D-CF screen. This success rate could

even be increased to 75% by using the more efficient CECF

Figure 6. Characterization of CF produced YedZ. (A) Coomassie Blue stained samples of 10 mL expressed YedZ derivatives after IMAC. (B)

SEC profiles of purified apo-YedZ produced in different CF expression modes. (C) CD spectroscopy of apo-YedZ. The a-helical content was

calculated to 55% and the melting point was determined at 661C. (D) UV–vis spectroscopy of holo-YedZ. Oxidized holo-YedZ has a single

absorption maximum at 412 nm including free hemin at 401 nm. Cofactor specific absorption at 425, 528 and 558 nm is detected when the

sample is reduced with sodium dithionite.

Table 3. CF production of holo-YedZ

Expression Detergenta) Cofactorb) Elutionc) A280 Ratio A280/A445 SECd)

D-CF (Brij-78) Brij-78 Apo 1.41 90.29 111

P-CF (LMPG) DDM Apo 1.53 n.d. 111

D-CF (Brij-78) DDM Holo (c/p) 1.59 2.43 �

DDM Holo (c) 1.61 20.14 11

P-CF (LPPG) DDM Holo (p) 1.56 1.8 11

(DPC) DDM Holo (p) 1.61 1.42 �

(SDS) DDM Holo (p) 1.52 1.78 1

Controle)

D-CF (Brij-78) Brij-78 Holo (p) 1.17 169.3 1

DDM Holo (c) 1.37 61.4P-CF (LMPG) DDM Holo (p) 1.4 16.25 1

a) Detergent used for analysis.b) Cofactor incorporation: c, co-translationally; p, post-translationally.c) Peak maximum in mL.d) Peak shape after size exclusion chromatography: �, apparent aggregation; 1, irregular peak shape, 11, mostly regular peak shape, 11

1, ideal peak shape.e) Proline transporter PutP.

Proteomics 2010, 10, 1762–1779 1775


configuration. The results obtained by GFP monitoring

were verified in the subsequent CE-P-CF screen imple-

menting SDS-PAGE analysis and immunoblotting of the

expressed MPs. This correlation demonstrates that, despite

significant restrictions have to be considered, GFP still

can be used as reliable reporter of MP expression in suitable

D-CF approaches.

The CF MP expression was systematically analyzed in

three independent screens: (i) automated batch-D-CF

screen, (ii) CE-P-CF screen and (iii) CE-D-CF screen and the

estimated yields were rated into four levels.

Our batch protocol closely resembles the ‘‘Cytomim’’ system

which has been used for the throughput expression

screening of soluble proteins in microtiter plates before [25].

By the addition of components which energize the trans-

cription/translation process directly [26], expression yields of

15mg folded GFP in a 96-well V-shape plate at 321C in a

reaction volume of 25 mL could be achieved, corresponding

to �600 mg/mL of reaction. This is in accordance to

published efficiencies of batch reactions [12]. CECF proto-

cols can yield in up to several millilgrams of functional MPs

per single milliliter of reaction but are more difficult

to set-up by robotic platforms and they were thus

performed manually [6, 15]. Alternative CECF configura-

tions like microfluidic array devices are emerging and may

represent a future option for automatic throughput appli-

cations [27].

In the D-CF mode, the soluble expression of the smaller

targets having 300 amino acids is with 71% more efficient

than that of larger targets with 58%. However, as only the

soluble fraction of D-CF reactions were analyzed, the limited

range of analyzed detergents might account for this fact.

This result is so far only representative for using Brij-78 as

detergent, although among classical detergents such as

DDM, digitonin, Triton X-100 and others, Brij derivatives

are usually superior in their general solubilization proper-

ties in D-CF reactions [8]. However, the evaluation of newly

emerging detergents or hydrophobic compounds such as

fluorinated surfactants or amphipols would be important for

expanding the applications of the D-CF expression mode

[28]. Alternatively, providing hybrid micelles, bicelles, lipids

or modified expression kinetics might further be rational

approaches to modulate the efficiencies of co-translational

MP solubilization [10, 29]. A total of 48 (38%) targets are

produced in the two upper expression levels, including 19

MPs exceeding 300 amino acids. Protein size is the most

evident parameter that correlates with soluble expression.

Smaller MPs are clearly more likely targets, although a

number of exceptions such as AtoE, AmpE, ManZ or YbiP

have been identified. Hence, size is not an exclusive criter-

ion and it has to be considered that the screens were

performed with identical reaction protocols which will

represent non-optimal conditions, e.g. in ion concentrations,

hydrophobic environment or template design for a number

of the analyzed individual targets. It is thus very likely that

yields could significantly be improved by further optimizing

target dependent reaction protocols individually. Modifica-

tions of prime parameters like template design, DNA quality

as well as ion concentrations can have a significant impact

on the CF expression efficiencies [30].

Interestingly, in the P-CF mode size appears not as

limiting for efficient expression and the bias between the

expression of smaller (77%) and larger (72%) MPs are

almost negligible. About 53% of the targets were instantly

expressed in preparative levels approaching or exceeding

1 mg/mL of reaction. The higher success rate if compared

with the CE-D-CF screen indicates that numerous MPs are

synthesized but not solubilized in the D-CF mode. For

resolubilization of P-CF produced MPs, a variety of deter-

gents including LMPG, LPPG, DDM or OG can be efficient

[31]. Solubilization of precipitates into functional protein

after P-CF expression was shown for a variety of targets as

the multidrug transporter EmrE, the mechanosensitive

channel MscL and the eukaryotic organic ion transporters

OCT1/OCT2 and OAT1 [30]. Sample homogeneity as well as

specific oligomer formation after SEC was observed in

several other cases [6, 7, 31, 32]. Other MPs like the

nucleoside transporter Tsx and the human endothelin B

receptor show high sample quality and activity only after

D-CF expression [8, 33]. Both modes are therefore important

tools for the production of high quality samples and there-

fore simultaneous evaluation should be approached.

The expression of 22 targets (16%) was not detected in

any screen. Accumulation of rare codons can be excluded

and three targets showed even high expression in E. colicells [13]. The production of three targets (AbgT, FliF and

NhaB) appears to inhibit the CF system to some extend.

As none of these targets could be detected even by immu-

noblotting, it seems unlikely that the synthesized proteins

themselves might have some toxic effects. We rather

speculate that the observed inhibitory effects might be due

to interactions of premature translation products with

essential compounds of the expression machinery like the

ribosomes. Interestingly, these three MP-GFP fusions could

also not be produced in vivo [13]. All targets have been

synthesized without addition of any artificial N-terminal

tags. Formation of unfavorable mRNA secondary structures

which could prevent an efficient initiation of translation

might therefore be a more frequent reason for the failure of

expression [4, 34]. Modifications of the N-terminal end of

the targets by addition of small tags (e.g. His-, T7-, Strep-

tag), may improve success of translating the transcription

product [8, 34].

In summary, the expression of 112 targets representing a

total of 84% could be detected in at least one of the three

screens. This is particularly remarkable if compared with

common success rates of approaches in cellular systems

where usually the expression of not more than 30% of the

targets is monitored [31, 35, 36]. In addition, once a protocol

is established the CF production of MPs takes only 12 h and

samples can instantly be loaded on purification columns

without any cell disruption or membrane extraction steps



that are required with conventional cellular expression

protocols. The total costs for a preparative scale 1 mL CF

reaction can be calculated to approximately 60h, which is

competitive to most other conventional expression systems.

It should further be highlighted that CF expression offers

exceptional labelling opportunities for proteins in any

desired amino acid combination [37]. Besides supple-

mentation of labelled amino acids, no further protocol

optimization is required in order to achieve high efficiency

and complete label incorporation [38]. The total molecular

mass of a MP target plays a similar role for success in vivo.

Targets exceeding 50 kDa and having more than seven

TMSs have some 10% likelihood to become expressed [35].

An obvious correlation to codon usage was also not found.

Some specific functional classes of MPs belonging to

transporter families appear to be underrepresented in high

expressing targets which might be due to toxic conse-

quences resulting from biological functions of the MPs.

Furthermore, C-terminal GFP moieties will only mature invivo if the C-terminus of the target protein remains intra-

cellular, which is roughly the case for 70% of all MPs [20].

This limitation is eliminated in CF systems and we have

similar success rates of targets with proposed Cin and Cout

topologies. The expression of our MP-fusion library was

previously analyzed in E. coli cells by whole-cell GFP and/or

PhoA activities and the synthesis of 104 targets of our screen

was observed by this approach [13, 22]. In total, 22 of the 134

selected targets (16%) were exclusively obtained by CF

expression while vice versa, 14 (10%) targets have only been

identified by in vivo expression. CF and in vivo expression

therefore complement each other to a total success rate of

94% (126 targets). A similar complementary effect of CE-P-

CF expression and in vivo expression in E. coli cells was

previously reported for a selection of smaller MP targets in

sizes up to 30 kDa with a strong bias to hypothetical MPs

[31]. Our target list has an overlap of 21 targets with this

work and both combine to the CF expression profiling of

233 MPs of the E. coli proteome. Similar to our results, a

complementation of CF and in vivo expression to 75%

success was obtained. Most results nicely correlate in

between the two expression screens and few differences are

most likely due to different template designs of individual

targets.

Quality control of MP samples is a central issue for

further structural and biochemical analysis. In particular the

functional folding of cofactor containing MPs may be harder

to obtain. The putative flavocytochrome YedZ was produced

without GFP fusion in large-scale reactions in yields of

1.2 mg/mL after IMAC purification, demonstrating that

protocols obtained in analytical scale screens can rapidly be

scaled up. The resolubilization of P-CF produced YedZ in

the presence of the cofactors hemin and FMN resulted in

sample qualities comparable to YedZ samples isolated from

E. coli cells. Cofactor specific absorption maxima in depen-

dence from the oxidized or reduced state of holo-YedZ

correspond to previously published spectra of heme b

containing holo-YedZ [22]. This result is a further demon-

stration that CF expression allows the functional production

of even complex MPs containing non-covalently bound

cofactors and it also exemplifies an approach for the

modulation of cofactor incorporation by production of heme

containing holo-YedZ.

Key distinctions in between cellular and CF expression

systems is the exceptional versatility of the latter one in

offering a wide array of options to modify the expression

environment by choosing different expression modes and

implementing a rapidly increasing panel of detergents,

lipids or ligands. In this study, the non-ionic detergent

Tyloxapol of the group of alkyl aryl polyether alcohols was

first used to efficiently solubilize even large MPs in the

D-CF mode even in concentrations exceeding 100�CMC.

The zwitterionic detergent SB3-14 was first used to resolu-

bilize MPs after P-CF expression. Besides triggering

expression yields, modifications of the CF expression

environment are clearly prime determinants for the result-

ing quality of the produced MP samples as exemplified for

YedZ, NhaA, PutP and MtdB. Similar expression mode

dependent variations in sample homogeneity as well as in

functional properties of MPs have already been observed

previously [8, 33]. In practice, the CF expression of new MP

targets should be approached in two levels. First, an efficient

production protocol should be established either in the P-CF

mode or in the D-CF mode by taking advantage of appro-

priate GFP fusions. Main optimization parameters would be

template design and final Mg21 and K1 ion concentrations.

In the second level, the established expression protocol

would be taken as basis for sample quality optimization by

detergent screening in the P-CF and D-CF modes. For the

majority of expressed MPs of this report, so far no char-

acterization of sample quality has been done. The intention

of our screen was to show that many targets are easily

accessible and the delivered expression protocols could serve

as starting point for closer characterizations analogously to

the demonstrated models. In addition, further optimized

robotic programmes could be used for automatic quality

optimization of individual targets in 96-well formats

implementing a systematic evaluation of distinct reaction

compounds.

The authors are grateful to Birgit Sch.afer for excellent tech-nical assistance. The authors further thank Gunnar von Heijne,Michael Karas and Achim Hannappel for their support andhelpful discussions. The work was supported by the SFB 807‘‘Transport and Communication across Biological Membranes’’.

The authors have declared no conflict of interest.

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Cell-free expression profiling of E. coli inner membrane proteins