Tuning microbial hosts for membrane protein production.

Abstract

The last four years have brought exciting progress in membrane protein research. Finally those many efforts that have been put into expression of eukaryotic membrane proteins are coming to fruition and enable to solve an ever-growing number of high resolution structures. In the past, many skilful optimization steps were required to achieve sufficient expression of functional membrane proteins. Optimization was performed individually for every membrane protein, but provided insight about commonly encountered bottlenecks and, more importantly, general guidelines how to alleviate cellular limitations during microbial membrane protein expression. Lately, system-wide analyses are emerging as powerful means to decipher cellular bottlenecks during heterologous protein production and their use in microbial membrane protein expression has grown in popularity during the past months.This review covers the most prominent solutions and pitfalls in expression of eukaryotic membrane proteins using microbial hosts (prokaryotes, yeasts), highlights skilful applications of our basic understanding to improve membrane protein production. Omics technologies provide new concepts to engineer microbial hosts for membrane protein production.

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BioMed Central
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Microbial Cell Factories
Open Access
Review
Tuning microbial hosts for membrane protein production
Maria Freigassner1, Harald Pichler1,2 and Anton Glieder*1,2
Address: 1Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria and 2Austrian Centre for
Industrial Biotechnology (ACIB), c/o Research Centre Applied Biocatalysis, Petersgasse 14, A-8010 Graz, Austria
Email: Maria Freigassner - maria.freigassner@gmail.com; Harald Pichler - harald.pichler@tugraz.at; Anton Glieder* - glieder@glieder.com
* Corresponding author
Abstract
The last four years have brought exciting progress in membrane protein research. Finally those
many efforts that have been put into expression of eukaryotic membrane proteins are coming to
fruition and enable to solve an ever-growing number of high resolution structures. In the past, many
skilful optimization steps were required to achieve sufficient expression of functional membrane
proteins. Optimization was performed individually for every membrane protein, but provided
insight about commonly encountered bottlenecks and, more importantly, general guidelines how
to alleviate cellular limitations during microbial membrane protein expression. Lately, system-wide
analyses are emerging as powerful means to decipher cellular bottlenecks during heterologous
protein production and their use in microbial membrane protein expression has grown in
popularity during the past months.
This review covers the most prominent solutions and pitfalls in expression of eukaryotic membrane
proteins using microbial hosts (prokaryotes, yeasts), highlights skilful applications of our basic
understanding to improve membrane protein production. Omics technologies provide new
concepts to engineer microbial hosts for membrane protein production.
Background
We have seen amazing advances in the field of membrane
protein research over the past four years. For the first time,
high resolution structures for pharmaceutically relevant
eukaryotic membrane proteins, including class A G-pro-
tein coupled receptors (GPCR) [1-5], transporters [6] and
channel proteins [7-16] became available and provided
valuable insight into their mode of action (Table 1). A few
years earlier, structures of the soluble domains of human
CYP3A4 and CYP2D6 of the Cytochrome P450 family, the
most important drug metabolising enzymes, had been
solved [17-20]. Actually, many years of strenuous efforts
were required to get the proteins to crystals, relying on
iterative optimization of all steps from expression to puri-
fication and crystallization. The total number of mem-
brane protein structures deposited in the protein data
bank (PDB) also strikingly reflects that membrane protein
research can not yet keep pace with the one of soluble pro-
teins. Among over 58,000 total entries, only some 100 of
all coordinate sets belong to membrane proteins [21].
Nature, however, provides plenty of different targets:
roughly one third of all open reading frames encode for
membrane proteins, as predicted for fully sequenced
genomes of eubacterial, archaean and eukaryotic organ-
isms [22,23]. The discrepancy between biodiversity and
poor structural knowledge can be largely attributed to the
low natural expression of membrane proteins, to their
hydrophobic character which complicates overexpression
of functional membrane proteins, as well as to difficulties
during their purification and crystallization.
Published: 29 December 2009
Microbial Cell Factories 2009, 8:69doi:10.1186/1475-2859-8-69
Received: 15 October 2009
Accepted: 29 December 2009
This article is available from: http://www.microbialcellfactories.com/content/8/1/69
© 2009 Freigassner et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Table 1: Eukaryotic membrane proteins with high resolution structures.
ProteinExpression Host PDB coordinates Resolution (Å)Reference
TRANSMEMBRANE PROTEINS: BETA-BARREL
Beta-Barrel Membrane Proteins: Mitochondrial Outer Membrane
Human VDAC-1 voltage dependent anion channel
E. coli
2K4T1, 2JK42
4[210,211]
Murine VDAC-1 voltage dependent anion channel
E. coli
3EMN 2.3 [212]
TRANSMEMBRANE PROTEINS: ALPHA-HELICAL
G Protein-Coupled Receptors
Rhodopsin: Bovine Rod Outer Segment mutant N2C/D282CCOS cells2J4Y 3.4 [122]
Engineered turkey β1 adrenergic receptor
Trichoplusia ni
2VT4 2.7[1]
Human β2 adrenergic receptor, from β2AR365-Fab5 (2R4R) and
β2AR24/365- Fab5 complexes (2R4S)
Spodoptera frugiperda
2R4R, 2R4S
3.4/3.7[2]
Engineered human β2 adrenergic receptor
Spodoptera frugiperda
2RH1, 3D4S2.4/2.8[3,4]
Human A2A adenosine receptor, In complex with a high-affinity
subtype-selective antagonist ZM241385.
Spodoptera frugiperda
3EML 2.6[5]
Integrin Adhesion Receptors
Human Integrin αIIbβ3 transmembrane-cytoplasmic heterodimer,
NMR Structure
E. coli
2KNC[213]
Snare Protein Family
Syntaxin 1A/SNAP-25/Synaptobrevin-2 Complex from Rattus rattusE. coli
3HD73.4[214]
Ion Channels
Kir3.1-Prokaryotic Kir Chimera: Mus musculus & Burkholderia
xenovorans
E. coli
2QKS2.2[7]
ASIC1 Acid-Sensing Ion Channel: Gallus gallus; 2QTS: N- and C-
terminal deletions, 3HGC: minimal functional channel
Spodoptera frugiperda
2QTS, 3HGC1.9/3.0[8,215]
ATP-gated P2X4 ion channel (apo protein): Danio rerio (zebra fish)
(expressed in SF9 cells), 3.1 Å; Closed state. A construct, 3.5 Å:
3I5D
Spodoptera frugiperda
3H9V, 3I5D3.1/3.5[216]
Kv1.2 Voltage-gated potassium Channel: Rattus norvegicusPichia pastoris
2A79 2.9[9]
Kv1.2/Kv2.1 Voltage-gated potassium channel chimera: Rattus
norvegicus
Pichia pastoris
2R9R2.4[10]
Other Channels: Aquaporins and Glyceroporins
Rat AQP4 aquaporin water channel, S180D mutant (2ZZ9)
Spodoptera frugiperda
2D57, 2ZZ9 3.2/2.8[11,12]
Human AQP4 aquaporin water channel
Pichia pastoris
3GD81.8[13]
Human AQP5 aquaporin water channel
Pichia pastoris
3D9S2.0 [14]

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Paradoxically, from a pharmaceutical point of view, mem-
brane proteins - in particular GPCRs and transport pro-
teins - constitute key drug targets, as most signal
transduction processes are initiated or transmitted via
membrane proteins. The facts that at least 50% of all com-
mercially available drugs target membrane proteins, that
the major enzymes initiating drug metabolism are micro-
somal cytochrome P450 enzymes and that the human
immune defense against invading pathogens is often ini-
tiated by their membrane proteins [24,25], underline the
importance of elucidating three-dimensional structures of
membrane proteins [26]. Evidently, great efforts are made
to better understand membrane proteins from a structural
and functional point of view. Unfortunately, (recom-
binant) expression of eukaryotic membrane proteins
often suffered from low expression levels, instability of
proteins and/or degradation by the host's proteolytic
machinery. While in the past many labs have focused on
their single targets, there was a strong trend towards stud-
ying a large number of different proteins simultaneously
more recently. In this regard, many consortia and research
groups have tried to rationalize all processes from expres-
sion to purification and crystallization, hoping to end up
with at least a few membrane proteins that are amenable
to crystallization (structural genomics). Whatever route is
taken, it is obvious that improvements in early steps, par-
ticularly concerning expression levels of functional mem-
brane proteins are of tremendous benefit to subsequent
experiments, especially protein purification, characteriza-
tion and crystallization.
Review
1. Expression systems
In the past, unreasonably more structures have been
solved for bacterial and archaebacterial membrane pro-
teins than for eukaryotic ones (Database of Membrane
Proteins of Known Structure [21]). Early success in crystal-
lization of eukaryotic membrane proteins came from the
use of native proteins [27-31]. However, this was only suc-
cessful if a uniquely rich source was available, e.g. the ret-
Plant SoPIP2;1 aquaporin: Spinacia oleracea in closed (1Z98) and open
conformation (2B5F)
Pichia pastoris
1Z98, 2B5F
2.1/3.9 [15]
Pichia pastoris AQY1 aquaporin, pH 3.5 (2W2E) and 8.0 (2W1P)
Pichia pastoris
2W2E, 2W1P1.15/1.40[16]
Other Channels: Gap Junctions
Human Connexin 26 (Cx26; GJB2) gap junction
Spodoptera frugiperda
2ZW33.5[217]
Membrane-Associated Proteins in Eicosanoid and Glutathione Metabolism
Human Microsomal Prostaglandin E Synthase 1: Human (Electron
Diffraction) In complex with glutathione.
E. coli
3DWW3.5 [218]
Human 5-Lipoxygenase-Activating Protein (FLAP) with Bound MK-
591 Inhibitor (2Q7M), FLAP with iodinated MK-591 analog (2Q7R)
E. coli
2Q7M, 2Q7R
4.0[219]
Human Leukotriene LTC4 Synthase in complex with glutathione
S. pombe
2PNO3.3[220]
Human Leukotriene LTC4 Synthase in complex with glutathione/apo
form
Pichia pastoris
2UUH, 2UUI2.15/2.0[67]
ATP Binding Cassette Transporters
Mus musculus P-Glycoprotein (3G5U), With bound QZ59-RRR
(3G60) and QZ59-SSS (3G61)
Pichia pastoris
3G5U, 3G60, 3G61
3.8/4.4/4.35[6]
P-type ATPase
Human Na,K-ATPase Regulatory Protein FXYD1
E. coli
2JO11
[221]
Phospholamban homopentamer: Human sarcoplasmic reticulum
E. coli
1ZLL1, 1FJK1, 1FJP1
[222]
Plasma Membrane H+-ATPase: Arabidopsis thalianaS. cerevisiae
3B8C3.6[223]
1 Structure determined by NMR spectroscopy,
2 Structure determined by combining X-ray and NMR data.
Table 1: Eukaryotic membrane proteins with high resolution structures. (Continued)

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ina providing mg amounts of rhodopsin, and it precluded
protein modifications, e.g. isotope labelling for NMR
analysis. Due to difficulties in isolating native membrane
proteins in sufficient quantity and quality, strong efforts
were made towards establishing heterologous expression
systems for the production of eukaryotic membrane pro-
teins. Prior to 2005, no high resolution structure had been
solved for any recombinant eukaryotic membrane pro-
tein. Since then, 28 unique structures have been eluci-
dated for eukaryotic membrane proteins (Table 1).
Among microbes, the prokaryote E. coli and the yeast
Pichia pastoris have been most successfully used in produc-
tion of eukaryotic membrane proteins for structural anal-
yses.
1.1. Prokaryotic expression systems and their limitations in hosting
eukaryotic membrane proteins
E. coli still tops the list of the most popular expression sys-
tems for prokaryotic and eukaryotic membrane proteins,
given the broad choice of molecular biology tools and
strains available [32]. Its ease of handling, especially in a
high-throughput way, enabling analyses of many proteins
in parallel, sophisticated genetic manipulation tech-
niques, the availability of membrane protein-adapted
strains, but also cost effectiveness - all these factors con-
tribute to the popularity of E. coli and its preferential use
in structural genomics initiatives targeting membrane
proteins of both prokaryotic and eukaryotic origin. While
E. coli provides an optimal environment for prokaryotic
proteins, its capability to host eukaryotic membrane pro-
teins was limited, however [26,33]. Although reasonable
success in providing eukaryotic membrane proteins for
structural analyses has been documented (Table 1), suc-
cess rates are low compared to eukaryotic expression
hosts. Often, prokaryotic homologues are studied in lieu
of eukaryotic membrane proteins. However, only 13% of
all membrane protein families are common between
prokaryotes and eukaryotes [34].
For eukaryotic proteins, prokaryotic folding pathways
might be far from optimal, as subtle differences between
assembly machineries impede their correct processing.
While the eukaryotic translocon possesses charged resi-
dues mediating interaction with the nascent polypeptide
chain during translocation and insertion into the mem-
brane, bacterial counterparts are lacking these features
[35]. Additionally, the membrane composition of differ-
ent eukaryotic organellar membranes is different from the
prokaryotic cytoplasmic membrane and also processing
of premature proteins often fails in prokaryotes, likely
leading to mistargeting or misfolding of eukaryotic mem-
brane proteins [36]. Apart from incompatibilities in pro-
tein processing, varying polypeptide elongation and
folding rates contribute to the obstacles experienced in
heterologous expression of eukaryotic membrane pro-
teins in prokaryotic hosts [37]. The rate of polypeptide
chain elongation is 4 to 10 times faster in prokaryotes
compared to eukaryotic cells. As all steps during transla-
tion, folding and membrane insertion need to be properly
balanced allowing the protein to find its native conforma-
tion, high translation rates might lead to exposure of
hydrophobic segments, and thus promote their aggrega-
tion. In the past, membrane protein expression was per-
formed just like expression of simple soluble proteins.
Strong promoter systems have been preferentially
employed to drive expression, e.g. T7 promoter, thereby
obviously exceeding the translocation machinery's ability
to correctly process the enzyme [38]. As a consequence of
high transcript abundance combined with high prokaryo-
tic translational rates, eukaryotic membrane proteins
often accumulate in inclusion bodies [26,39]. At first
view, this offers some apparent advantages such as no or
reduced toxicity caused by heterologously expressed pro-
teins, protection against proteases and, thus, high yields.
However, inclusion bodies require renaturation, which is
anything but trivial. Although some membrane proteins
have been successfully refolded from inclusion bodies
[40], in vitro renaturation remains a contentious issue, as
many proteins fail in reaching the native, functional con-
formation once they are denatured. Refolding has been
most successfully employed for β-barrel type membrane
proteins, which are innately more robust than α-helical
membrane proteins, but also not as sought after as the lat-
ter [41]. Therefore, expression of properly folded and
membrane-embedded proteins is highly desirable, be it
by attenuating expression strength, e.g. by the use of weak
promoters and low copy number, be it by modifying cul-
tivation conditions, e.g. growth temperature and inducer
concentration, as will be discussed further below [38,42].
In the case of low temperature, the effect of reduced tran-
scription and translation rates seems to combine with the
availability of additional chaperones and foldases [43].
Overexpression of eukaryotic proteins is furthermore
often complicated due to E. coli's inability to perform
posttranslational modifications that can be crucial for
proper folding, targeting and function [44]. Many eukary-
otic membrane proteins rely on the presence of specific
post-translational modifications (PTM) to attain full
activity, e.g. acylation and phosphorylation, or to retain
stability mainly by glycosylation [33]. E. coli is not able to
glycosylate proteins and, thus, not the host of choice if
such modification is crucial for activity [33,44,45]. On the
other hand, non-glycosylated proteins are usually rather
homogeneous and uniform, which might be advanta-
geous for experiments depending on very pure enzymes,
e.g. crystallization. Often, phosphorylation events are
necessary for protein activation e.g. triggering conforma-
tion changes as observed for many GPCRs, thus preclud-
ing in vivo studies [46,47]. Further, E. coli lacks

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endogenous G proteins impeding its use for functional
studies of GPCRs, but coexpression [48] or even external
addition of the G protein α subunit to the membrane
preparation was able to restore high-affinity binding
properties [49].
According to the current view, membrane proteins exist as
protein-lipid complexes with specific lipids inevitably
required to promote folding, retain activity and confer
structural stability, as can be seen from their presence in
many crystal structures [36,50,51]. Bacterial membranes
are devoid of sterols and derivatives, polyunsaturated fatty
acid chains and sphingolipids, which are known to play a
key role in folding of membrane proteins in eukaryotic
membranes (reviewed in [36]). Failure to produce func-
tional mammalian receptors in E. coli has thus been partly
ascribed to the lack of essential lipids, e.g. cholesterol dur-
ing expression of mammalian serotonin transporter
[52,53]. Many mitochondrial membrane proteins tightly
interact with cardiolipin, and large amounts of F1F0 ATP
synthase were only obtained after modulating lipid bio-
synthesis pathways and, thus, lipid composition of E. coli
membranes towards an increased cardiolipin content
[54]. In a few cases, the addition of lipids during expres-
sion and purification restored protein activity [36].
Although reasonable expression levels have been demon-
strated for different individual eukaryotic membrane pro-
teins [42], the most successful attempts have been found
for β-barrel type membrane proteins (Table 1). Expression
levels were typically several orders of magnitude lower
compared to their bacterial homologs [52].
Besides E. coli, the gram-positive bacterium Lactococcus lac-
tis has emerged as alternative prokaryotic host due to a rel-
atively small number of endogenous membrane proteins
and thus high capacity to host foreign proteins [55-58]. As
facultative anaerobic bacterium, L. lactis is able to reach
high cell densities even under anaerobic conditions. Being
surrounded by a single membrane, L. lactis cells can be
broken easily, thereby facilitating downstream processes.
Due to the relatively small genome i.e. approx. 50% of E.
coli, however, some auxiliary proteins and chaperones
with essential functions in folding and membrane inser-
tion might not be provided by L. lactis [57]. While 21 of
25 (84%) prokaryotic transport proteins were successfully
expressed in E. coli, only 10 thereof (40%) could be
obtained in L. lactis cells, as demonstrated recently [59].
On the other hand, the proportion of functional protein
of prokaryotic origin was higher in L. lactis compared to E.
coli [60]. Though levels of eukaryotic membrane proteins
lag still behind those obtained for prokaryotic ones, yields
are comparable to those obtained in E. coli [57]. In light
of the fact that formation of inclusion bodies has not been
observed so far during expression of membrane proteins,
L. lactis provides an attractive alternative to E. coli [55,57].
The halophilic archaeon Halobacterium salinarum also
ranges among promising non-standard expression hosts
due to its elaborate membrane system and, thus, its high
capacity to host additional membrane proteins. Nonethe-
less, high level expression seemed to be limited to bacteri-
orhodopsin and derivatives [61,62]. Among prokaryotic
hosts, the photosynthetic bacteria Rhodobacter sphaeroides
and Rhodobacter capsulatus provide plenty of membrane
space while concomitantly offering elaborate and highly
efficient insertion machineries. During prototrophic
growth, they use light for energy production and growth,
and thereby enrich intracytoplasmic membranes [63-65].
However future experiments will have to demonstrate
whether this is sufficient to obtain high yields of mem-
brane proteins.
1.2. Yeasts as expression systems for eukaryotic membrane proteins
Despite individual breakthroughs, prokaryotic expression
systems tend to provide an inhospitable environment for
eukaryotic membrane proteins. Therefore, most recent
attempts were directed towards employing eukaryotic
hosts for eukaryotic membrane protein expression. In this
context, yeasts are an inexpensive, yet efficient alternative
to prokaryotes. Providing a eukaryotic environment, yeast
cells are capable of processing proteins similarly to higher
eukaryotes. Simple handling, rapid growth and powerful
genetic tools contributed to their popularity [66]. To date,
10 out of 28 unique structures of heterologously
expressed eukaryotic membrane proteins were obtained
from yeast-derived material, including the best-resolved
structures (1.8 - 2.0 Å) of mammalian membrane proteins
(Table 1) [13,14,67].
Most commonly, membrane proteins are overexpressed
and targeted to the ER membrane from where they are
transported to the plasma membrane in vesicles [68].
Unlike E. coli, yeasts are able to perform various post-
translational modifications including proteolytic process-
ing of signal sequences of both pre- and prepro-type,
disulfide-bond formation, acylation, prenylation, phos-
phorylation and certain types of O- and N-linked glyco-
sylation that all might be essential for activity and correct
folding (reviewed in [66]). Methylotrophic yeasts are an
attractive alternative to S. cerevisiae as overexpressed pro-
teins are less frequently hyper-mannosylated than in S.
cerevisiae. In addition, the presence of a terminal α-1,2-
mannose residue abolishes their allergenic potential
[69,70]. Meanwhile, even strains with a more uniform
and also with a human glycosylation pattern are available
for functional studies [71-74]. Although the lipid compo-
sition of yeast membranes is closer to higher eukaryotes
than to E. coli, the lack of certain lipids, in particular ster-

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ols, e.g. cholesterol for mammalian proteins, sito-, stigma-
and campesterol for plant enzymes, might affect protein
functionality. Ergosterol, the predominant endogenous
sterol in fungi, might compensate for specific sterols, as
shown for mammalian GPCRs and transport proteins, but
for full activity presence of cholesterol might be essential
[33,75]. During purification, protein activity and stability
can be retained or even restored by addition of defined
lipids. In this respect, cholesterol hemisuccinate was cru-
cial in stabilizing the A2a receptor during purification fol-
lowing overexpression in S. cerevisiae [76].
Being the model eukaryote, S. cerevisiae has traditionally
been most widely employed for the expression of eukary-
otic membrane proteins [77-79]. This popularity can be
largely ascribed to elaborate, yet easy genetic manipula-
tion techniques, as documented by the availability of
numerous expression plasmids. Furthermore, the vast
number of available strains including entire deletion
libraries allows to carry out functional complementation
in vivo, as exemplified recently for different plant trans-
porter proteins [80-82]. Most frequently, inducible pro-
moters (e.g. GAL1 or GAL10) are employed to tune
membrane protein expression by yeast as for 93% of
membrane proteins constitutive expression resulted in
impaired growth during their homologous overexpres-
sion [83,84]. In contrast to other yeasts, protocols have
been established for S. cerevisiae to enable its use in high-
throughput (HTP)-expression studies of eukaryotic mem-
brane proteins [77-79]. While on the one hand a C-termi-
nal GFP-His8-fusion is employed to assess expression
levels and optimize purification [77,78], other methods
rely on the combination of C-terminal His10- and N-ter-
minal FLAG-tags [79], using immunodetection for estima-
tion of expression levels. Most commonly, episomal
plasmids were used for expression. Ability of S. cerevisiae
to perform in vivo recombination allows ligation of the
respective gene into the expression plasmid in vivo [77-
79]. Additionally, in vivo cloning permits the simple con-
struction of protein variants, as shown recently for mouse-
TRPM5 channel [85], and, therefore, directed evolution of
membrane proteins or selected domains.
Due to its tendency to hypermannosylate proteins and
complex handling of S. cerevisiae in fermenter cultures,
much attention has recently focused on non-conventional
yeasts including Schizosaccharomyces pombe, Yarrowia lipol-
ytica, Hansenula polymorpha and Pichia pastoris.
The fission yeast Schizosaccharomyces pombe outperforms
other yeast species in expressing mammalian proteins
with mammalian-like core glycosylation pattern and by
its ability to process intron-containing genes [86,87].
Thus, S. pombe is a promising, yet easy-to-handle alterna-
tive to conventional yeast species with a high potential for
the expression of complex proteins, e.g GPCRs [88]. S.
pombe's suitability to express functional eukaryotic mem-
brane proteins was supported by a previous study on
human D2S dopamine receptor, resulting in a five fold
higher binding affinity of isolated membranes and, thus,
a higher proportion of functional, membrane-integrated
receptor compared to the protein produced in S. cerevisiae
[89].
Frequently, heterologous systems are used for membrane
protein expression to obtain sufficient amounts of those
proteins for structural and functional studies in vitro.
Apart from purification-oriented purposes, eukaryotic
membrane proteins such as enzymes of the cytochrome
P450 family are also overexpressed in heterologous sys-
tems to perform whole-cell biotransformations on highly
valuable substances (reviewed in [90]). While initially
bioconversions had been realized using S. cerevisiae [91-
97], meanwhile also S. pombe has been successfully
employed for mammalian cytochrome P450-mediated
reactions [98-102]. As P450s act in concert with an elec-
tron-donating system, coupling efficiency and thus sub-
strate conversion rates can be increased by coexpression of
the respective mammalian redox partners or by protein
engineering of the biocatalyst [103]. The origin and the
quantity of the coexpressed reductase may influence the
expression of the heme domain and the resulting activity
of these complex enzymes. This is usually regulated by
gene copy numbers or by the choice of the employed pro-
moters.
Similarly, another non-conventional yeast species, Yar-
rowia lipolytica, is emerging as host for biotransforma-
tions of hydrophobic substrates as it tolerates organic
solvents and metabolizes aliphatic compounds. Y. lipolyt-
ica has been employed for production of cytochrome
P450 enzymes, including bovine P450 17α (CYP17A)
[104], human P450 CYP1A1 [105] and CYP53B1 from
Rhodotorula minuta [106]. From these studies evidence
emerged that integration of multiple expression cassettes
had a positive effect on heterologous expression as
observed by increased conversion rates.
Among non-Saccharomyces yeasts, the methylotrophic
yeast Pichia pastoris has been most extensively and suc-
cessfully employed in membrane protein research (Table
1). Exceptionally high cell densities can be reached during
cultivation, concomitantly guaranteeing efficient and eco-
nomically sustainable protein production (reviewed in
[107,108]). Methylotrophic yeasts may derive all energy
for growth from the utilization of methanol as carbon
source by inducing expression of methanol-metabolizing
enzymes, namely alcohol oxidase (AOX) and dihydroxy-
acetone synthase (DHAS) [109]. Because promoters of the
methanol utilization pathway are strong but also tightly

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regulated, these are most commonly employed to drive
heterologous protein expression [110,111], allowing to
reach up to 90 mg L-1 of membrane proteins exemplified
by a human aquaporin [112]. Furthermore, protein pro-
duction capacity is not compromised during growth on
minimal media, which makes this yeast attractive for the
production of stable isotope labelled proteins for NMR-
based structural biology [113] and also for reliable indus-
trial production. Apart from well-known advantages for
protein expression, such as eukaryotic proteolytic process-
ing, protein folding, disulfide bond formation and glyco-
sylation, another factor contributing to Pichia pastoris
outstanding popularity is its ability to secrete heterolo-
gous proteins while secreting very low levels of endog-
enous proteins. This allows extracellular accumulation of
heterologously expressed soluble proteins, but also offers
a route for production of eukaryotic membrane proteins
as well, as they are efficiently trafficked through ER and
Golgi to the plasma membrane.
Versatility of Pichia pastoris as host for membrane protein
expression has been documented by the number of struc-
tures elucidated with Pichia-derived material (Table 1). In
particular, a success rate of 93,5% has been found for
expression of GPCRs, whereas more than half of all 100
proteins tested failed to be properly expressed in E. coli
[26]. A closer analysis of specific binding activities also
demonstrated that Pichia pastoris can keep up with higher
eukaryotic systems, e.g. Semliki Forest virus-mediated
expression in mammalian cells, but also showed that the
ideal host is target dependent.
Thus, using complementary hosts allows to expand the
expression space. If eukaryotic membrane proteins fail to
be properly expressed in yeasts, attention is usually drawn
to higher eukaryotic hosts, e.g. insect cells, mammalian
cells or cell-free expression systems [114-116]. These
expression hosts are not covered by this review, but are
outlined in detail in recent articles[26,117,118].
2. Strategies for membrane protein overexpression and
problems encountered therein
Like for soluble proteins, each membrane protein behaves
in an individual, unfortunately unpredictable way upon
overexpression. In previous studies, no correlation
between the "expressability" of a membrane protein and
protein specific parameters, i.e. size, number of trans-
membrane helices, hydrophobicity, was found during
homologous overexpression of 300 membrane proteins
in E. coli ([119], reviewed in [120]). Other studies pointed
out that heterologous expression of GPCRs in E. coli is
limited to a size of 54 kDa and below [26]. Similarly, in S.
cerevisiae, one of the most significant factors affecting the
expression level was considered to be the molecular size
of the protein - smaller proteins (< 60 kDa) generally tend
to be better expressed than larger ones, as judged from
homologous overexpression of more than 1000 mem-
brane proteins in S. cerevisiae [84]. White and coworkers
also found an inverse correlation between the number of
transmembrane helices and the expression level, but pres-
ence of large extramembraneous domains might amelio-
rate a membrane protein's performance [84]. Highest
expression levels were obtained for proteins with less than
five transmembrane segments - preferentially composed
of hydrophobic residues and lacking charged amino acids.
Recently, in a "discovery-oriented" selection process, 234
out of 384 endogenous integral membrane proteins rep-
resenting every IMP Pfam family within the yeast genome
were successfully expressed under control of the GAL1
promoter in S. cerevisiae corresponding to a success rate of
61% with expression levels ranging from 0.5 to 5.8 mg L-
1 [79]. Furthermore 25% of all candidate proteins were
amenable to purification using n-Dodecyl-β-D-maltoside,
as judged from immunoblotting of membrane extracts
and the protein's performance during size-exclusion chro-
matography. Like in previous studies, the success rate for
expression of integral membrane proteins was tightly
linked to their specific properties. Best expression levels
were obtained for small proteins (MW < 100 kDa) with a
low number of transmembrane helices (<6) and a low
average hydrophobicity index [79]. For heterologous pro-
teins comparative studies are missing. However, a general
trend was that proteins perform better during heterolo-
gous expression when related homologous proteins are
present in the respective expression host [121]. However,
most strategies towards optimization of membrane pro-
tein expression focused on modifying the protein itself
and optimizing cultivation conditions rather than engi-
neering the expression host.
2.1. Engineering membrane proteins for increased stability, affinity or
activity
Improvements in expression levels can also be achieved
by modifying the membrane protein. For the purpose of
crystallization, the protein structure can be stabilized by
increasing rigidity, e.g. through introduction of disulfide
bonds [122] or N- and/or C-terminal truncations [11].
Furthermore, target proteins may be tailored by incorpo-
ration of point mutations with the aim to destroy biolog-
ical activity and, thus, to diminish toxicity during
heterologous overexpression [123]. In this regard,
directed evolution strategies have turned out to be more
straightforward compared to rational engineering, as the
latter relies on structural information which is still scarce
for membrane proteins. By employing directed evolution,
expression levels, stability and binding selectivity of deter-
gent-solubilized membrane proteins have been improved
[124,125]. Variants of the turkey β1-adrenergic receptor
with improved conformational homogeneity in presence
of antagonist, enhanced tolerance to short-chain deter-

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gents and thermostability have been obtained by alanine
scanning followed by in-depth mutagenesis and combi-
nation of hot spots in E. coli [126].
To date, screening for improved variants or enhanced
expression level has been almost exclusively carried out in
E. coli, by employing immunodetection (colony-filtration
blot of membranes) [127], FACS in the presence of spe-
cific fluorescent ligands [125] or specific activity [124]
and ligand binding assays [126,128]. Like for soluble pro-
teins, activity-based screens allow to select for functional
mutants [124], while screens relying on total expression
levels including all immunodetection-based assays might
preferentially enrich inactive, hence non-toxic proteins.
Use of membranes instead of whole cells in combination
with activity assays can circumvent the specified troubles,
as suggested by Molina and coworkers [129]. Following
initial screening using E. coli, selected variants are trans-
ferred to eukaryotic systems for production purposes
[125]. Besides their impact on protein stability and func-
tion, mutations might directly affect transcriptional,
translational and/or folding efficiency. In respect to differ-
ences in cellular milieus in between species, many prom-
ising candidates might fail in providing the same
properties they have been selected for during the initial
screening in prokaryotes. Recently, the suitability of S. cer-
evisiae to perform directed evolution experiments has
been demonstrated, by improving ligand sensitivity of a
human UDP-glucose receptor [130]. Meanwhile, the
availability of protocols paves the way for the use of vari-
ous yeast species as hosts in directed evolution experi-
ments [85,131].
2.2. Adaptation of the host to membrane protein production
2.2.1. Relieving metabolic stress evoked by membrane protein
overexpression by adjusting transcription and translation efficiency to
membrane protein folding
Apart from engineering proteins of interest, expression of
membrane proteins can be fine-tuned on different cellular
levels in order to improve yields. During production of
membrane proteins high gene dosage strategies often fail
in providing more functional protein but rather entail
impaired growth or accumulation of aggregated protein in
inclusion bodies as a result of limited cellular capacity to
correctly translocate, assemble, modify and integrate pro-
teins into the membrane [39,132-135]. As transcript
abundance does not limit membrane protein production
[136], assembly of membrane proteins often works better
when synthesis rates are slowed down to better match the
rates of insertion and assembly. Frequently, a high copy
number entails an increase in total yield, but not in
amounts of functional protein, as a consequence of
improper balance of protein biosynthesis and folding
[135,137,138]. Low transcriptional rates can be achieved
by lowering the gene dosage or switching to weak, yet
tightly regulated promoters, thereby avoiding accumula-
tion of aggregated protein [38]. As a consequence of mem-
brane protein production, cell growth is often retarded,
thus inducible promoter systems may be superior to con-
stitutive ones in membrane protein production (e.g. PBAD
and PT7 for E. coli, PAOX1 for Pichia pastoris), as they allow
unimpaired cell growth to high densities prior to protein
expression. Apart from low gene dosage or weak promot-
ers, expression kinetics can be fine-tuned by shortening
induction periods, reducing inducer concentrations or
switching to weak inducers, e.g. lactose for the T7 pro-
moter [132,139,140].
If transcription efficiency limits the final yield, use of well-
expressing genes in bicistronic expression constructs
might be exploited to increase mRNA levels [141]. Effi-
ciency of transcription initiation can be mediated by
adapting the codon usage of the 5' region of the recom-
binant gene to the host's own one, but can also be
improved by fusing well-expressed genes, encoding for
e.g. β-galactosidase upstream of the gene of interest [142].
Another way to guide the protein along the secretory route
to the plasma membrane is the use of N-terminal, host-
specific secretory targeting sequences, e.g. of the Ste2
receptor [88,143], the prepropeptide of the S. cerevisiae α-
mating factor or the signal peptide of acid phosphatase
[26,135-138,144-149]. The majority of integral mem-
brane proteins (IMPs) however possesses a native N-ter-
minal signal sequence, thus the use of N-terminal tags -
either for detection by immunoblotting or for purification
- might lead to delusive conclusions due to processing of
the signal peptide [79]. When information on protein
topology is scarce, the use of N-terminal secretory signal
sequences might also interfere with protein folding and
insertion into the membrane. This strategy, however, usu-
ally worked for GPCRs, where the N-terminus is facing the
outward side of the membrane [150]. Apart from signal
sequences, also N- or C-terminal fusions with soluble
cytoplasmic (GFP, glutathione S-transferase) and peri-
plasmic proteins (maltose binding protein) may improve
expression of otherwise poorly expressed targets and pro-
mote their stabilization in E. coli, putatively by protecting
membrane proteins against proteolytic degradation of
vulnerable N- or C-termini [151-155]. In this context, tar-
geting to and integration into bacterial membranes has
also been facilitated by fusion to other membrane pro-
teins (maltoporin, glycerol-conducting channel GlpF,
mistic) [142,156]. Due to its ability to integrate into the
membrane independently of the translocon, mistic, an
integral 13 kDa membrane protein derived from Bacillus
subtilis, can be used to eliminate bottlenecks encountered
therein [157,158]. Evidently, topologies of both mem-
brane proteins - target protein and fusion partner - have to
be considered, e.g. the N-terminal fusion of GlpF is lim-

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ited to membrane proteins with N-termini facing the cyto-
plasm [156].
Finally, external parameters considerably affect transcrip-
tion and translation rates, cultivation temperature being
recognized as the most effective one. Generally, best
growth conditions in terms of biomass yield are not nec-
essarily ideal for heterologous protein expression, but
might rather elicit a stress response [159] or result in low
yield of functional membrane protein [144,160]. In fact,
folding and membrane insertion of the protein can be
promoted by lowering the cultivation temperature
[140,144,149,160,161]. At low temperature the activity of
the transcriptional and translational apparatus is reduced,
avoiding an overload thereof. Additionally, many cold-
shock chaperones are induced at lower temperature,
which promotes folding [162], but also reduces proteo-
lytic degradation [163,164]. The positive effect might fur-
ther be linked to higher protein stability at low
temperatures, but also to decreased folding stress. Espe-
cially in prokaryotes, folding of eukaryotic membrane
proteins benefits from reduced transcription and transla-
tion rates as a result of adaptation to eukaryotic transla-
tion rates.
Effects of other external parameters, i.e. pH, osmolarity
and aeration, on membrane protein expression have not
been studied thoroughly so far. Only the influence of pH
on the yield of individual membrane proteins has been
documented for yeast, suggesting optimal values ranging
from neutral to alkaline pH [160,161]. These studies can
not serve as general guidelines, however. Optimization is
required for each target protein.
2.2.2. Relieving folding and translocation stress
The gross majority of membrane proteins follow the same
route like soluble, secretory proteins once the nascent
polypeptide emerges from the ribosome, including target-
ing to the translocon via the SRP particle (Figure 1, Figure
2). In contrast to secretory proteins, however, the most
prominent bottleneck in overexpression of membrane
proteins is the physical space within or at a lipid bilayer.
Difficulties arise from limitations in membrane capacity
when accommodating additional proteins. Each mem-
brane has an optimal ratio between lipids and membrane
proteins. Thus, variations caused by massive protein inser-
tion affect membrane integrity and cell functionality
[165]. Upon disturbance of this balance, expression sys-
tems often react with stress responses including protein
degradation.
Prokaroytes
In prokaryotes, the SRP guides a nascent polypeptide
chain to its membrane-associated receptor FtsY and subse-
quently to the Sec translocon consisting of the protein
conducting channel SecY, SecE and SecG [166]. Sec trans-
locon-mediated insertion into the cytoplasmic membrane
is supported by the peripherally associated ATPase SecA,
which is required for translocation of periplasmic loops
and secretory proteins, and by YidC, which is thought to
mediate lateral transport of transmembrane helices
(TMH) into the membrane (Figure 1). Two proteases
residing in the cytoplasmic membrane, FtsH and HtpX,
play a crucial role in quality control of membrane pro-
teins [167].
During membrane protein production, enhanced levels of
folding proteins might be required in order to cope with
the membrane protein load. As a shortage of foldases
might limit the cellular productivity, membrane protein
production can be optimized by providing surplus com-
pounds of the assembly machinery. The effect of coexpres-
sion and deletion of chaperones and proteases has been
recently investigated for E. coli expressing GPCRs
[168,169]. Using the human cannabinoid receptor CB1 as
model protein, a transposon library of E. coli was screened
for increased expression levels [169]. Inactivation of DnaJ
(dnaJ::Tn5) resulted in a higher abundance of membrane-
integrated receptor and also abolished growth-retarding
effects of overexpression. On the other hand, coexpres-
sion of DnaJ/K during expression of the magnesium trans-
porter CorA increased expression levels, and its role in
membrane protein assembly has been ascribed to its capa-
bility to prevent accumulation of the protein in inclusion
bodies [170]. From X-ray structural analysis, evidence
emerged that presence of a large extramembraneous
domain requires an increased amount of chaperones to
mediate its folding [171]. The exact role of DnaJ during
CB1 expression is not clear. According to topology predic-
tions, the receptor lacks any large loops connecting its 7
TMHs. However, the N-terminal 116 amino acids were
found to be located on the extracellular side, while the
shorter C-terminal domain faces the cytoplasm. It has
thus been speculated that the inhibitory role of DnaJ is
linked to its ability to interact with the N-terminus pro-
ceeding from the ribosome, thus preventing its insertion
into the Sec translocon [169]. Given that only total
amounts of receptor are indicated, it remains to be clari-
fied, whether non-functional and, thus, non-toxic protein
has accumulated in the membrane due to absence of fold-
ing chaperones or whether the positive effect of dnaJ
knockout is due to its strong interaction with the N-termi-
nal region. Apart from dnaJ, positive effects on membrane
protein biogenesis have also been observed upon inacti-
vation of the transcription factor nhaR, which regulates
expression of genes involved in cation transport and of
the DNA damage-inducible helicase dinG. Yet, their pre-
cise role during membrane protein biogenesis need to be
elucidated in detail [169]. Judging from the expression

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yields obtained for other membrane proteins (human
CB2 receptor, human bradykinin receptor 2, human neu-
rokinin receptor 1), the length of the N-terminal tail
seems to play a crucial role, as no improvement has been
achieved by transposon-mediated dnaJ inactivation.
Another study addressed the effect of coexpression of dif-
ferent chaperones, translocon compounds and proteases
on membrane protein biogenesis, exemplified for the cen-
tral and peripheral cannabinoid receptor (CB1 and CB2),
the bradykinin receptor 2 (BR2) and the neurokinin-(sub-
stance 1) receptor 1 (NKR1) [168]. While many chaper-
ones including the membrane integrase YidC, SRP
compound 4.5S RNA, SRP receptor FtsY, compounds of
the translocon SecY and SecE and the chaperones SecB,
GroEL/GroES did not promote membrane protein pro-
duction, a moderate increase in expression levels of CB1
has been achieved by coexpression of the SRP compound
Ffh, chaperones DnaJ/K and the peptidyl-prolyl isomerase
trigger factor Tig - thus opposing the previous study [169].
Unexpectedly, highest expression levels were obtained
upon coexpression of FtsH, a membrane-anchored AAA+
protease [169]. Besides improved amounts of membrane-
inserted receptor, a two-fold higher cell density provided
Membrane protein biogenesis in prokaryotes
Figure 1
Membrane protein biogenesis in prokaryotes. In prokaryotes, most membrane proteins are targeted to and inserted
into the cytoplasmic membrane by the SRP pathway, which involves interaction of the growing polypeptide chain with the sig-
nal recognition particle (SRP) and its receptor FtsY, binding of the nascent polypeptide chain-ribosome-complex to the
SecYEG/YidC pore, translocation of cytoplasmic and periplasmic loops across the cytoplasmic membrane and their folding by
SecA and various chaperones and insertion of hydrophobic segments into the membrane. The autonomous, YidC and Tat
pathways, that are used by small proteins and membrane-associated periplasmic proteins, respectively, are mentioned here for
sake of completeness.

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Figure 2 (see legend on next page)

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evidence that cells experienced less stress, although cell
division was impaired as shown by cell elongation. The
fact that no improvement in binding efficiency of GPCRs
was achieved and strains reached higher cell densities
compared to control strains left a few questions unan-
swered. FtsH is involved in quality control of membrane
proteins, however no effect has been observed for other
AAA+ proteases (HtpX, YcaL, YfgC, and YggG). Prelimi-
nary results of a transcriptome analysis indicated that
glycerol metabolism was largely rearranged in mutant
strains, suggesting that the lipid composition of cytoplas-
mic membranes was altered [168]. Recent studies exam-
ined the stress response upon CB1 or FtsH production and
revealed an induction of genes from the σ32 heat shock
regulon [172]. Using promoter-GFP fusions, it was dem-
onstrated that coexpression of CB1 and FtsH resulted in
additive stress responses as assessed by flow cytometry,
clearly indicating that FtsH is not able to attenuate stress
responses caused by CB1 expression. Instead, stress-
related effects were boosted further by FtsH coexpression.
Authors proposed that this protease likely proactively pre-
pares the cells to cope with the toxic effects caused by CB1.
Eukaryotes
Typically, membrane proteins - excluding proteins des-
tined for peroxisomal, chloroplast and mitochondrial
membranes - enter the secretory pathway by translocating
into the ER membrane where folding and maturation of
the proteins take place [68,173,174]. Like in prokaryotes,
as soon as the signal sequence protrudes from the the
growing polypeptide-ribosome complex, the SRP guides
the complex to the ER membrane (Figure 2). Translation
resumes when the complex docks to the Sec61p pore, trig-
gering opening of the lumenal BiP-mediated gate. Yeast
cells deploy several mechanisms to cope with the
increased membrane protein load. Overproduction of ER-
resident membrane proteins triggers enhanced prolifera-
tion of ER membranes as shown for HMG-CoA reductase
and CYP52 A3, mediated by induction of the unfolded
protein response [175-177]. At the same time, ER chaper-
ones act in concert to properly assemble membrane pro-
teins or promote folding of lumenal, extramembraneous
domains, thus their coexpression can boost ER folding
capacity. Previously, expression levels of a serotonin trans-
porter in the baculovirus expression system were
improved by a factor of three upon coexpression of the
ER-resident chaperone calnexin [178]. As this membrane
protein requires the presence of a distinct N-glycan moiety
to mature into functional protein, increased levels of cal-
nexin were required to ensure efficient N-glycosylation
within the ER. Coexpression of calreticulin and BiP also
increased the final yield, albeit to a lesser extent. Similarly,
increased abundance of the dnaK-type chaperone BiP
mRNA has been found during overexpression of cyto-
chrome P450 52A3 in S. cerevisiae [176,177] and of the 2-
compound cytochrome P450 system including P450
reductase and benzoate p-hydroxylase BphA in A. niger
[179]. On the other hand, deletion of Cne1, a yeast
homolog of mammalian calnexin and calreticulin,
resulted in higher levels of human transferrin receptor in
S. cerevisiae [180]. As part of the ER quality system, cal-
nexin was thought to prevent transport of the human
transferrin receptor to the plasma membrane, which was
then reversed by CNE1 deletion. Furthermore, increased
levels of Sec61 translocon have been observed during
membrane protein production [177]. Folding of trans-
membrane segments is largely mediated by membrane-
resident chaperones, which often display a high substrate
specificity. Folding of amino acid permeases, hexose
transporters, phosphate transporters and chitin synthase
III was facilitated by chaperones Shr3, Gsf2, Pho86 and
Chs7 respectively [181-183]. Deletion of the respective
chaperone resulted in protein aggregation and failure of
proteins to exit the ER [182].
Besides mediating membrane protein folding, the ER
serves as a quality checkpoint to control protein integrity
before the protein exits the ER. If cellular capacities to cor-
rectly assemble membrane proteins are exhausted, aber-
rant protein accumulates within the ER and is subjected to
different rescue or degradation routes. Eukaryotic cells
Membrane protein biogenesis in eukaryotes
Figure 2 (see previous page)
Membrane protein biogenesis in eukaryotes. In eukaryotic cells, membrane protein biogenesis occurs in a cotransla-
tional way. Proteins residing in membranes of ER and Golgi apparatus or in plasma membrane use the secretory pathway. Like
in prokaryotes, SRP recognizes polypeptides protruding from the ribosome complex, thereby transiently attenuating transla-
tion. As soon as the SRP-ribosome complex interacts with the SRP receptor and docks to the Sec61 translocon pore, transla-
tion resumes, BiP relocates, thereby opening the lumenal gate and the membrane protein enters the membrane by lateral
diffusion through the Sec61 pore. Peroxisomal membrane proteins either use the Pex19/Pex3-mediated way (class I proteins,
shown here) or are thought to reach the peroxisomal membrane via the ER (class II) [208]. Mitochondrial membrane proteins
pass through the outer mitochondrial membrane (OMM) via the TOM complex (translocase of the outer membrane). While β-
barrel proteins of the OMM are transported to the SAM and MDM complexes (sorting and assembly machinery), the TIM22
and TIM23 complexes (translocase of the inner mitochondrial membrane) are used to target proteins to the inner mitochon-
drial membrane [209].

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employ different strategies to cope with the accumulation
of misfolded proteins within the ER. First, inhibition of
translation initiation counteracts further accumulation of
unfolded polypeptides. Secondly, under conditions of
increased membrane protein load, cells overcome folding
limitations by upregulating expression of chaperones,
thereby increasing the folding capacity within the ER
[184]. The increased chaperone requirement in turn often
triggers induction of the UPR, an intracellular signalling
pathway that acts to relieve stress situations [185,186].
Finally, proteins that fail to reach their native fold are tar-
geted to the proteasome for degradation, through disloca-
tion to the cytoplasm and protein ubiquitination.
Depending on the topology and location of the misfolded
domain, different pathways are employed for membrane
protein turnover. While proteins with large cytosolic
domains enter the Doa10-dependent route, lumenally
exposed lesions target the protein to the Hrd1 degradation
pathway [187-191].
Being an indicator for folding problems and ER stress, the
UPR has also been rationally exploited as a sensing mech-
anism allowing optimized production of functional
membrane proteins in S. cerevisiae [186]. The trypano-
somal H+/adenosine cotransporter was used as model
protein and β-galactosidase as reporter with a transcrip-
tional UPR element driving expression of the lacZ gene.
Expression conditions were optimized for the transporter
by minimizing UPR activation. This strategy, however,
only works for membrane proteins that elicit the UPR
upon overproduction as observed for evolutionarily diver-
gent proteins, while most proteins from more closely
related organisms, e.g. yeast receptor and plant transport
proteins, did not highlight any folding problems.
2.2.3. Increasing protein stability by chemical chaperones
Membrane proteins often associate with specific com-
pounds in vivo, be it defined lipids or interacting proteins,
and their presence is crucial for proper folding, activity
and/or stability. In this context, the subcellular localiza-
tion plays a significant role. Many mitochondrial mem-
brane proteins tightly interact with cardiolipin, as shown
by their association in protein crystals even after the pro-
tein has been subjected to several purification steps
[192,193]. Thus, shortage or even lack of defined lipid
molecules may affect heterologous expression of mem-
brane proteins. Lipid compositions vary between different
hosts and organelles (reviewed by [36]). For full activity,
it is often crucial to keep the membrane protein in a sim-
ilar environment as in its native host, e.g. by targeting it to
the proper organelle or by choosing a related host. Mem-
brane proteins may assemble into supercomplexes in vivo
[194]. Thus, co-expression of proteins that are known to
form stable complexes or tightly interact with the target
protein might also improve overexpression. For example,
the coexpression of human β2-adrenergic receptor and its
corresponding mammalian G protein subunit in S. cerevi-
siae did not only improve stability, but was also necessary
for in vivo functionality [143]. Similarly, small chemical
ligands can reduce conformational flexibility by tight
association, and thus decrease the likelihood of hydro-
phobic domains to be exposed and attacked by proteases
or becoming prone to aggregation. This idea has been suc-
cessfully applied in heterologous expression of GPCRs. By
addition of specific ligands at concentrations close to 100
times the Kd value during the induction phase, expression
levels and stability of
[135,144,149].
GPCRs were increased
Apart from specific ligands, chemical supplements, e.g.
DMSO, glycerol and histidine, are known to affect mem-
brane protein expression positively as evaluated exten-
sively for GPCRs [135,144,195]. The stimulating effect of
DMSO at low concentrations (2.5% (v/v)) seems to be
linked to its ability to modify the physical properties of
membranes and membrane capacity by upregulating tran-
scription of genes involved in lipid biosynthesis [196].
DMSO also increases the permeability of membranes and,
thus, improves the access of externally added ligands to
membrane proteins [197]. Like for many chaperones, use
of DMSO is not a universal key to success, as adverse
effects including downregulated expression and proteoly-
sis have also been observed for plasma membrane trans-
porters in yeasts [198]. Chaperoning activities and
stabilization of protein conformation, especially of glyco-
proteins, have been ascribed to glycerol. Addition of 10%
(v/v) glycerol during growth led to an increase in expres-
sion levels, exemplified for a human P-glycoprotein dur-
ing heterologous expression in S. cerevisiae [195]. Glycerol
was thought to directly increase stability of the membrane
protein. The positive influence of histidine, which is most
often added at concentrations of 0.04 mg mL-1, is thought
to be related to its ability to act as a physiological antioxi-
dant, but the trivial possibility of improved availability of
this amino acid can not be ruled out [199].
2.2.4. Rationalizing membrane protein production by engineering
host cells
As membrane protein biogenesis requires a tight balance
between different processes and even small changes might
have far-reaching consequences the application of gener-
ally valid engineering approaches is complicated. In the
past, attention had been chiefly drawn to modifications of
cultivation conditions or of expression constructs, i.e.
gene dosage, promoters, purification tags, leader
sequences, fusion proteins, in order to optimize mem-
brane protein production. If expression failed in an
expression system a different expression host was investi-
gated for its ability to correctly synthesize the protein.
Experience has shown that many years of effort are neces-

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sary in order to succeed in protein crystallization. Many
improvements have been achieved on a trial-and-error
basis. Unfortunately, they did not provide any general
guideline for membrane protein production, not to men-
tion any insights into the physiological constraints host
cells encounter. There are no great endeavours to adapt
microbial expression hosts to membrane protein produc-
tion. Instead, many labs have resorted to well-established
protease-deficient strains in order to minimize proteolytic
degradation rather than alleviating host-specific bottle-
necks [77,79,144].
Low success rates and protein aggregation have sparked
interest in the mechanisms and physiological effects of
membrane protein biogenesis allowing identification of
putative bottlenecks. Analysing the consequences of
membrane protein production from a comprehensive
molecular and cell biological view - accomplished on the
transcriptome and/or proteome level - allows to study
physiological effects, cellular constraints and stress regula-
tion. Most importantly, the -omics approach will help to
apply the compiled knowledge for rational engineering of
the host strain. Although no general, single-key bottle-
necks in membrane protein expression are known so far,
factors like chaperones, foldases, proteases etc., which
were found to facilitate membrane protein expression in
single cases, are in focus of comprehensive molecular
analyses of membrane protein overexpressing hosts by
transcriptomics or proteomics. Several groups have
already addressed the question whether genetic engineer-
ing of the host, going beyond simply shifting the rate-lim-
iting step within one single metabolic pathway or process,
can relieve specific bottlenecks.
2.2.4.1. Classical engineering
The traditional way to improve heterologous hosts in
terms of productivity is the classical strain engineering,
involving chemical mutagens or UV radiation. In fact, so
far only E. coli BL21(DE3) has been tailored for improved
membrane protein production capacity by classical selec-
tion more than one decade ago [39]. After having recog-
nized that overexpression of an oxoglutarate-malate
carrier protein negatively affected cell growth, strains that
were able to survive during protein expression were
selected, termed Walker strains or C41(DE3) and
C43(DE3). Interestingly, in these strains higher expres-
sion levels and unimpaired growth were observed upon
overexpression of soluble and membrane proteins that
otherwise hampered cell viability of BL21(DE3). Miroux
and Walker also noted that lower transcript levels had
been reached by the mutant strains compared to the
BL21(DE3), suggesting that transcriptional rates had
slowed down. Later, it was postulated that the high suc-
cess rate of Walker strains was linked to their ability to
proliferate additional intracellular membranes and, thus,
tolerate increased membrane protein loads [54]. In order
to decipher the mechanisms, which make these strains
superior to conventional ones for membrane protein pro-
duction, the proteome of the Walker strains overexpress-
ing the prokaryotic membrane protein YidC was
compared to the one obtained from the parental strain
BL21(DE3) [38].
Although the Walker strains behaved superior regarding
growth characteristics and final expression levels, the
same proteome response was observed in all strains, thus
giving no clue what was the key to success in the Walker
strains. However, lower abundance of chaperones and
proteases (ClpB, IbpA, HslUV) involved in resolving
cytosolic aggregates indicated that in contrast to
BL21(DE3) the Walker strains experienced fewer prob-
lems in folding and insertion of membrane proteins at the
Sec translocon. Driven by many open questions, the de
Gier group analyzed the genetic features of the strains in
detail and discovered that the high success rate of the
Walker strains in membrane protein expression likely
originates from a lower transcriptional rate caused by
reduced abundance of T7 RNA polymerase, which is used
in these strains to drive recombinant protein expression.
Mutations in the lacUV5 promoter, a strong variant of the
wild-type lac promoter driving expression of the T7 RNA
polymerase, have accumulated in these strains which
decrease the promoter strength. As a result, transcription,
translation and protein insertion into the membrane are
well balanced. Cells more easily cope with the membrane
protein load and accomodate them in a functional way
into the cytoplasmic membrane [38]. The mutations lead-
ing to lower expression rate accumulated in the -10 region
thereby reconverting the lacUV5 variant to the wildtype
lac promoter, and in the lac operator region making the
promoter susceptible to catabolite repression by glucose.
The latter resulted in delayed expression. In turn, the de
Gier lab has constructed engineered strains, designated
Lemo21(DE3), which allow to control activity of T7 RNA
polymerase by its inhibitor T7 lysozyme (T7Lys). In these
strains, coexpression of T7Lys under control of the titrable
rhamnose inducible promoter rhaBAD is exploited to
rationally dampen T7 RNA polymerase activity. This strat-
egy allows to fine-tune expression of soluble and mem-
brane-embedded protein of both prokaryotic and
eukaryotic origin in E. coli by avoiding conditions which
result in aggregation of protein and recruitment of chap-
erones and proteases. Once again this study demonstrated
that slowing down transcription is one key to success for
membrane protein biogenesis.

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2.2.4.2. The use of -omics technologies to rationalize
membrane protein production
Prokaryotic systems
Motivated by poor knowledge on the host's physiological
response to membrane protein production, Wagner and
coworkers systematically analysed the effect of overex-
pression of three different membrane proteins (YidC,
YedZ, LepI) on the proteome of E. coli [200]. Overexpres-
sion of all model proteins hampered growth, resulting in
early transition to stationary phase. According to flow
cytometry, cells were slightly bigger, indicating increased
proliferation of additional membranes to host foreign
membrane proteins, but also impaired cell division.
Other studies have also pointed out that E. coli cells over-
come physical limitations by proliferating additional
membranes in order to cope with the increased mem-
brane protein load [201]. Judging from the proteome of
cell lysate, protein aggregates and cytoplasmic mem-
branes, it became clear that massive expression of mem-
brane proteins severely disturbed protein homeostasis in
the cytoplasm of E. coli [200]. In production strains, over-
loading of the translocon entailed an accumulation of
cytoplasmic aggregates, thereby evoking sequestration of
chaperones like DnaJ/K and GroEL/S as shown by immu-
noblotting. Increased abundance of proteolytic proteins
(HslU/V, ClpXP, ClpB; assisting proteins IbpA/B) com-
bined with higher levels of chaperones suggested that in
response to membrane protein misfolding the heat shock
reponse is activated. The recruitment of chaperones and
proteases was required to reduce inclusion body forma-
tion and resolve overexpressed protein that had accumu-
lated in inclusion bodies. As revealed by transcriptome
analysis, transcription of heat shock genes (lon, ClpP,
HslU/V) and molecular chaperones (DnaK, DnaJ) was
upregulated in response to inclusion body formation
[202,203].
Besides the overexpressed membrane proteins, cytoplas-
mic aggregates contained chaperones, proteases (HslU/V,
ClpXP), many essential cytoplasmic proteins and many
precursors of periplasmic and outer membrane proteins,
indicating that the capacity of the translocation machin-
ery was saturated [200]. Therefore, coupling of transcrip-
tion, translation and targeting needs to be properly
balanced. In this specific case, protein aggregation was
probably also evoked by the use of the strong promoter of
the T7 polymerase which was employed to drive expres-
sion of the membrane proteins. The idea that membrane
protein production exceeded the capacity of the Sec trans-
locon was confirmed by different observations. On the
one hand, secretion efficiency was reduced suggesting that
cotranslational membrane protein biosynthesis competed
with the posttranslational process of protein secretion.
On the other hand, low levels of endogenous membrane
proteins provided evidence that recombinant membrane
protein outcompeted endogenous protein production, i.e.
most prominently respiratory chain complexes. Increased
levels of translocon components SecY, SecE and SecG and
membrane-associated ribosomal subunit L5 however,
inferred a saturation of the translocation capacity. Mem-
brane protein overproduction compromised cellular res-
piration, as shown by reduced oxygen uptake rates and
low cytochrome c oxidase activity. As a consequence of
inefficient energy metabolism, the Arc two-component
system was activated and the acetate-phosphotransacety-
lase pathway was primarily employed for ATP production.
Interestingly, expression of all model proteins evoked
similar responses on the proteome level, suggesting that E.
coli faces similar bottlenecks during membrane protein
expression irrespective of the protein. This sounds feasible
as membrane proteins are typically overexpressed in the
plasma membrane in prokaryotes, while in eukaryotic
cells membrane proteins pass through and end up in dif-
ferent organelles and, thus, optimization approaches
depend on the subcellular localization. It should be
emphasized that depending on protein structure different,
translocon-independent pathways might be used for tar-
geting and membrane insertion in prokaryotes as
observed for the magnesium transporter CorA [171]. This
work also carries great promise for future host engineering
and holds out the prospect that obstacles encountered in
membrane protein production can be alleviated on a
broad basis and do not require optimization for each indi-
vidual protein. Based on the existing data, compounds of
the translocon machinery are promising candidates for
cell engineering with the aim to obtain higher productiv-
ity. Although not yet demonstrated, the authors also sug-
gested the FtsH protease as putative target for knockout or
inhibition. Alternatively, coexpressing FtsH inhibitors, e.g
the peptide SpoVM derived from Bacillus subtilis, might
improve membrane protein overexpression. However,
this would conceptually contradict the unexpectedly pos-
itive effects of FtsH overexpression [169].
Yeast cells
The physiological response of yeast to membrane protein
production has hardly been deciphered in a rational way.
Some early studies aimed at considering membrane pro-
tein production in relation to cell physiology with the aim
to minimize folding stress within the ER by circumventing
induction of the UPR [186]. In order to reveal which fac-
tors govern membrane protein degradation and whether
ER-localized membrane proteins follow the ERAD degra-
dation route, a recent study analyzed the transcriptome of
S. cerevisiae cells overexpressing a non-functional variant
of the 12 TMH ABC transporter Ste6 [204]. Due to
improper folding of the extramembraneous cytoplasmic