Appl Microbiol Biotechnol (2013) 97:3811–3826
DOI 10.1007/s00253-013-4831-z
MINI-REVIEW
Optimisation of signal peptide for recombinant protein
secretion in bacterial hosts
Kheng Oon Low & Nor Muhammad Mahadi &
Rosli Md. Illias
Received: 5 January 2013 / Revised: 3 March 2013 / Accepted: 4 March 2013 / Published online: 26 March 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract Escherichia coli—the powerhouse for recombi-
nant protein production—is rapidly gaining status as a reli-
able and efficient host for secretory expression. An
improved understanding of protein translocation processes
and its mechanisms has inspired and accelerated the devel-
opment of new tools and applications in this field and, in
particular, a more efficient secretion signal. Several impor-
tant characteristics and requirements are summarised for the
design of a more efficient signal peptide for the production
of recombinant proteins in E. coli. General approaches and
strategies to optimise the signal peptide, including the se-
lection and modification of the signal peptide components,
are included. Several challenges in the secretory production
of recombinant proteins are discussed, and research ap-
proaches designed to meet these challenges are proposed.
Keywords Signal peptide . Recombinant protein .
Mutation . Secretory production . Sec system .
Escherichia coli
Introduction
Recombinant protein production processes in microbial
hosts are a major factor in modern biotechnology and bio-
based economies. The main obstacles in the production of
K. O. Low : R. Md. Illias (*)
Department of Bioprocess Engineering, Faculty of Chemical
Engineering, Universiti Teknologi Malaysia, 81310, Skudai, Johor
Bahru, Malaysia
e-mail: r-rosli@utm.my
K. O. Low
e-mail: khengoon@hotmail.com
N. Muhammad Mahadi
Malaysia Genome Institute (MGI), Ministry of Science,
Technology and Innovation, Jalan Bangi Lama, 43000, Kajang,
Selangor, Malaysia
recombinant proteins are low yields and inefficient purifi-
cation processes. The ability of many organisms to transport
native proteins away from the cytoplasm has sparked an
interest in developing a secretory production process for
recombinant proteins. Secretory expression offers many ad-
vantages when compared with cytoplasmic expression: it
simplifies the detection and purification of the product, re-
duces the complexity of the bioprocess, minimises the cell-
associated proteolytic degradation and improves the protein
folding and quality (Choi and Lee 2004).
As one of the most well-known microbes, Escherichia coli
is the most commonly used host for the production of recom-
binant proteins. Although not suitable for proteins that are rich
in cysteine residues or that require extensive post-translational
modifications, these bacteria offer the easiest, most econom-
ical, and fastest expression of heterologous proteins (Terpe
2006; Demain and Vaishnav 2009; Chen 2012). The features
of E. coli as a secretory expression host have been reviewed
extensively (Choi and Lee 2004; Mergulhao et al. 2005; Wong
et al. 2012), as have strategies to achieve high levels of
recombinant protein production (Yoon et al. 2010; Shokri et
al. 2003; Choi et al. 2006). This robust host has multiple
complex secretion pathways that are capable of exporting a
wide range of recombinant proteins when fused to specific
secretion signals. However, its secretion capacity is often
limited to a frustratingly low level compared to other expres-
sion hosts (i.e. Bacillus subtilis). Recent findings suggest that
the full potential of this microbe has yet to be fully explored
and realised. Here, we discuss several of the latest develop-
ments of this field, particularly as related to E. coli.
The hallmark of gram-negative bacteria is that the cell
envelope is composed of two membranes (the inner and
outer membranes), which are separated by a compartment
(the periplasm) that contains a thin peptidoglycan (or mu-
rein) layer. In E. coli, the export of protein is generally
accomplished by the Sec secretion system, also known as
the general secretory pathway, through one or both of the
3812
membranes. A target protein, fused to an N-terminal secre-
tion signal known as a signal peptide, can be recognised and
translocated by the Sec machinery to the periplasm. The
process of passing the protein through the inner membrane
and into the periplasm is called secretion or secretory pro-
duction. If the target protein further crosses the outer mem-
brane to the extracellular medium, the process is termed as
excretion or excretory production. On the one hand, gram-
positive bacteria, such as Bacillus, contain only a single cell
membrane with a thick peptidoglycan layer. In this case,
exported protein traverses only one membrane to reach the
extracellular medium and the process is called secretion.
Despite differences in their final destinations, the secretion
and excretion processes rely on the same Sec machinery to
traverse the inner membrane. The signal peptide plays a
direct and decisive role in directing the target protein to
the Sec machinery and across inner membrane. To achieve
a high level of protein secretion, the choice of signal peptide
remains the first and most important hurdle to be overcome,
but a comprehensive paper that reviews the state-of-the-art
in signal peptide optimisation is not available. This article
aims to complement the existing reviews by focusing on the
optimisation of the signal peptide, particularly for the selec-
tion and modification of the signal peptide for high levels of
recombinant protein secretion in E. coli. Relevant studies
that used signal peptide in other bacterial hosts, such as
Bacillus, are also included. Several aspects that were cov-
ered in other recent reviews are not discussed, including the
role of codon bias (Zalucki et al. 2009) and translation
(Zalucki et al. 2011) of the signal sequence in protein
export. Strategies to release recombinant proteins from the
periplasm to the extracellular medium include the use of cell
envelope mutants, co-expression of lysis promoting proteins
and manipulation of the culture conditions and medium (Li
et al. 2010; Sommer et al. 2010; Ni and Chen 2009). The
literature survey was performed using the PubMed database
and references therein.
Signal peptide and protein translocation across
the cell membrane
In E. coli, as much as 50 % of all cellular proteins are
exported, with more than 90 % translocated via the Sec
pathway. After emerging from the ribosome, the process of
secreting a protein involves a cascade of interactions with
the Sec machinery before reaching the final destination
(Fig. 1) (reviewed in Rusch and Kendall 2007). SecB binds
to the pre-protein, maintaining a transport-competent state
(Hardy and Randall 1991). SecA functions as a translocation
ATPase (Economou and Wickner 1994). Upon binding with
the pre-protein and adenosine triphosphate (ATP), SecA
inserts into the membrane (Ulbrandt et al. 1992), forming
Appl Microbiol Biotechnol (2013) 97:3811–3826
a translocation complex with the Sec translocase—
SecY/SecE/SecG (Georgiou and Segatori 2005). SecE and
SecY form a protein-conducting channel, with SecG stimulat-
ing the translocation activity. Proteins are translocated in an
unfolded state using energy from ATP hydrolysis and a proton
gradient (Papanikou et al. 2007). The translocation process is
completed after the signal peptidase cleaves the signal peptide
(Wickner et al. 1987) and SecD releases the mature protein
from the cytoplasmic membrane (Matsuyama et al. 1993).
The protein intended for secretion is routed to the
SecA/SecYEG complex in a SecB-dependent or signal recog-
nition particle (SRP)-dependent manner. SecB, or in some
cases DnaK (Mujacic et al. 2004), targets less hydrophobic
signal peptides—post-translationally—to the Sec translocase
(Papanikou et al. 2007). SRP targets highly hydrophobic signal
peptides and membrane proteins—co-translationally—to the
Sec complex. The SRP pathway includes the E. coli SRP (ffh),
a cytosolic ribonucleoprotein composed of 4.5S RNA and a
single polypeptide, and FtsY (Luirink and Dobberstein 1994).
SRP binds to the newly synthesised signal peptide, which then
targets the ribosome to the translocase via the FtsY receptor.
The secretion machinery differentiates secreted from
non-secreted proteins through recognition of the signal pep-
tide (Gouridis et al. 2009). Typically 15–30 amino acids
long, these amino-terminal pre-sequences are characterised
by their three distinct regions, a 5- to 8-residue-long posi-
tively charged N terminus (n-region), an 8- to 12-residue-
long hydrophobic core (h-region), and a polar 5- to 7-
residue-long carboxyl-terminal cleavage region (c-region)
(Fig. 2). Although all signal peptides contain these regions,
variability at the amino acid level is high. This variation
likely determines the capacity of the signal peptide to drive
protein secretion. By understanding the role of the different
amino acids at specific positions, signal peptides with an
improved ability to confer high levels of protein secretion
can be designed. Biochemical data (Mori et al. 1997) and
high-resolution nuclear magnetic resonance images of the
signal peptide-bound SecA (Gelis et al. 2007; Chou and
Gierasch 2005) have suggested several important consider-
ations and characteristics to be used in designing a function-
al signal peptide. For example, the signal peptide h-region
adopts an extended α-helical conformation that binds to the
SecA groove. In addition, the positively charged residues in
the n-region form salt bridges with the acidic residues adja-
cent to the SecA groove, while the c-region remains largely
exposed and without detectable interactions.
Another protein secretion system that utilises N-terminal
signal peptides is the Sec-independent twin-arginine trans-
port (Tat) system (Palmer and Berks 2012) (Fig. 1). In E.
coli, the Tat translocon consists of three different membrane
proteins, TatA, TatB and TatC (Muller 2005). TatBC com-
plex functions in the recognition of targeted proteins, which
triggers the recruitment of the TatA complex to form a
Appl Microbiol Biotechnol (2013) 97:3811–3826
Fig. 1 Schematic diagram of the major signal peptide-based secretion
system and process. A and B represent the Sec system, and C represents
the Tat system. A Membrane proteins and highly hydrophobic signal
peptides follow the co-translational translocation, or SRP, route. SRP
binds to an emerging nascent polypeptide chain to form an SRP–
nascent chain–ribosome complex. A receptor, FtsY, then binds to the
complex and guides it to the SecYEG translocon in the presence of
GTP. Once it has arrived at the SecYEG, the pre-protein enters the
membrane through the SecYEG gate/‘tunnel’ as it is being synthesised.
SecA provides mechanical work by hydrolysing ATP. B Most periplas-
mic proteins and less hydrophobic signal peptides follow the post-
translational translocation, or SecB, route. SecB binds to a largely or
fully synthesised secreted protein and maintains it in an unfolded state.
Cytoplasmic or SecYEG-bound SecA searches for the signal peptide
functional translocon (70 Å or more in diameter) within the
cell membrane (Muller 2005). In addition to the three-
distinct-domain architecture of the Sec signal peptide, Tat
signal peptides contain a few unique characteristics (Berks
et al. 2000): (1) an (S/T)-R-R-x-F-L-K sequence motif at the
n/h-region boundary, from which the twin arginine pathway
gets its name; (2) a relatively longer length and an extended
n-region; and (3) a less hydrophobic h-region, with a higher
occurrence of glycine and threonine residues than leucine.
Unlike secretion by the Sec pathway, which requires that the
protein remain in an unfolded state for export, the Tat
pathway recognises folded protein for export to the peri-
plasm. In general, proteins that have cofactors or fold rap-
idly in the cytoplasm can be transported along the Tat route.
3813
and guides the pre-protein through the SecYEG translocon, at the
expense of ATP. SecDF aids in the release of the pre-protein into the
periplasm. SPase I cleaves the signal peptide, releasing the mature
domain to the periplasm. Periplasmic chaperones may aid in protein
folding. C Rapidly folded proteins that harbour the Tat signal peptide
follow the Tat route. After being synthesised, the pre-protein folds
rapidly into its native conformation, sometimes with the help of cyto-
plasmic chaperones and cofactors. Tat protein is not recognised by the
Sec components, possibly because of the Tat signal sequence and/or
because of the rapidly folded nature of the pre-protein. TatBC recog-
nises the Tat protein and assembles the TatA translocon, through which
the Tat protein is traversed into periplasm. Similar to the Sec system,
SPase I (not shown) cleaves the Tat signal peptide and releases the
mature domain into the periplasm
Passenger protein: secreted or not secreted?
One of the pressing concerns in secretory recombinant pro-
tein production is understanding the characteristics of the
target protein that allow the secretion process to occur. This
remains a black-box issue and involves trial-and-error rather
than rational evaluation. It is generally believed that natively
secreted proteins are good candidates for secretory produc-
tion in heterologous hosts. However, studies have shown
that natively cytoplasmic proteins can also be secreted in
high levels when fused to a signal peptide. Some examples
include the secretion of winter flounder antifreeze protein
(Tong et al. 2000) and Manduca diuresin (Wong et al. 2003)
using the OmpA signal peptide in E. coli. For the Sec
3814
Fig. 2 General features of a Sec system signal peptide. a A typical
signal peptide contains three functional domains: a positively charged
n-region, a hydrophobic h-region and a polar c-region. A pro-region,
also known as the export initiation domain, is also found in some
secreted proteins. Signal peptidase I recognises and cleaves at the c-
region AxA motif (indicated by downward-pointing arrow). b A
summary of a more efficient signal peptide design. A net positive
charge in the n-region and an AAA codon at the second codon (P2).
The AAA codon permits strong translation initiation and codes for a
positively charged amino acid, lysine. A highly hydrophobic h-region
arising from a stretch of leucine amino acids and the presence of a helix
breaker residue, glycine, in the middle. An extended motif, xxAxA, for
the c-region, where x represents average-to-large amino acids, from
position −1 to −5. A net negative charge in the pro-region, with small
and negatively charged amino acids in the first two positions
pathway, one of the most important characteristics of the
secreted protein is that it has a slow folding rate or remains
in an unfolded state for a sufficient time to allow recognition
and translocation by the secretion pathway. If the target
protein folds rapidly or tightly in the cytoplasm, one may
opt to use a Tat signal peptide or engineer the target protein
to have slower folding kinetics. The latter approach was
shown to be feasible by Bakkes et al. (2010) for protein
exported by the type I haemolysin transport system in E.
coli. Similar to the Sec system, the haemolysin transport
system recognises and translocates unfolded polypeptides.
The authors created a ‘folding’ mutation, Y283D, of malt-
ose binding protein (MBP) and showed that the mutant was
translocated more efficient than its wild-type counterpart.
The approach may also be practical for the Sec pathway. On
the other hand, recent evidence has shown that proteins that
have undergone post-translational modification were equal-
ly well recognised and translocated through the Sec pathway
(Chen et al. 2012).
Optimisation of the signal peptide
Signal peptide selection
The signal peptide functions like a global positioning sys-
tem, providing the information needed to direct the target
protein to the final destination. After its synthesis in the
cytoplasm, a polypeptide competes with other transport-
competent polypeptides for export. As an analogy, one can
imagine a busy airport with only one take-off lane, limiting
Appl Microbiol Biotechnol (2013) 97:3811–3826
the amount of aircraft that can flow through the system.
Choosing a suitable signal peptide constitutes the first im-
portant step in constructing an extracellular recombinant
protein secretion system. For E. coli, the type of signal
peptide may be chosen based on the commercial availability
(i.e. PelB in a pET vector), a literature survey (i.e. MBP,
OmpA) or personal preferences. Signal peptides that have
previously performed well in E. coli may serve as good
candidates; several are listed in Table 1. With the limited
choices available in the literature, more efficient signal
peptides may need to be developed. Systematic approaches
in searching for the ideal natural signal peptide have been
developed in several laboratories. With ambitious genome
projects and sophisticated in silico prediction programs, the
identification of all signal sequences in a host cell can be
made and evaluated to enhance recombinant protein secre-
tion. Currently, the National Center for Biotechnology
Information (NCBI) hosts more than 1,000 genome se-
quences for a range of organisms, including several popular
bacterial expression hosts such as E. coli, Bacillus, and
Lactobacillus (ftp://ftp.ncbi.nih.gov/genomes/Bacteria).
Table 2 lists several of the commonly used computational
tools for signal sequence prediction. SignalP (Petersen et al.
2011) and Phobius (Kall et al. 2007) rank among the most
accurate and versatile tools that permit high-throughput
processing of protein sequences in a relatively short period
of time. A signal peptide library can be constructed by
cloning the signal-encoding nucleotides into an expression
vector, and these can be screened for protein production.
This approach has been proven feasible, as demonstrated by
several studies. For instance, Brockmeier et al. (2006) and
Degering et al. (2010) constructed libraries consisting of all
naturally occurring (non-lipoprotein) Sec-type signal se-
quences from B. subtilis (173 signal sequences) and
Bacillus licheniformis (220 signal sequences) and screened
them for heterologous protein secretion (cutinase from
Fusarium solani pisi and subtilisin from Bacillus
amyloliquefaciens). These studies revealed that signal pep-
tides can have large variations in production levels,
depending on the type of recombinant protein fused to them.
The optimal signal peptide for one protein was inefficient
for another protein and vice versa. A similar outcome was
obtained for a library of 76 Sec-type signal peptides from
Lactobacillus plantarum, which was screened for the effi-
ciency of protein secretion using staphylococcal nuclease
(NucA) and lactobacillal amylase (AmyA) as the reporter
proteins (Mathiesen et al. 2009). No correlation with the n-
region net charge, hydrophobicity level or length could be
identified for the high secretion performance signal pep-
tides. In another example, 108 signal peptides derived from
a 408-signal-peptide library from Corynebacterium
glutamicum R were evaluated for secretion efficiency using
active α-amylase from Geobacillus stearothermophilus
Appl Microbiol Biotechnol (2013) 97:3811–3826 3815

Table 1 Examples of several signal peptides that perform well in E. coli

Signal sequence Origin Recombinant protein Origin Yield Reference

Heat-labile E. coli Cholera toxin (ctxB) Vibrio cholerae 86–259 μg/ml (total of periplasm Jobling et al. (1997)
enterotoxin and supernatant)
LTIIb Alkaline phosphatase E. coli 0.1 U (1.7-fold greater than native
(phoA) phoA signal sequence under similar
expression conditions)
Pneumococcal surface Streptococcus Most abundant protein in periplasm
protein A (pspA) pneumoniae and supernatant
Alkaline E. coli Glucagon Human Up to 3.46 mg/L/OD600 at supernatant Wen et al. (2003)
phosphatase fraction
phoA Human epidermal Human 380 mg/L Zhang et al. (2007)
growth factor (hEGF)
Pectate lyase Erwinia Phospholipase A2 Streptomyces Up to 6 U/ml in the extracellular Takemori et al.
B PelB carotovora (PLA2) violaceoruber fraction; undetected in intracellular (2012)
fraction
Lipase Bacillus 35 % (U/g wet weight cell) in the Cho et al. (2000)
thermoleovorans extracellular fraction.
ompA E. coli Calcitonin peptide Salmon More than 200 mg/L in extracellular Ray et al. (2002)
(sCTgly) fraction when coexpressed with secE
and secY
GM-CSF Human Up to 0.7 g/l in soluble fraction under Sletta et al. (2007)
high cell density cultivation
Bordetella Avidin Chicken Up to 20 mg/L after purification from Hytonen et al.
avium periplasm (2004)
lamB E. coli Insulin-like growth Human Up to 8.5 g/L in the periplasm when Joly et al. (1998)
factor-I (IGF-I) coexpressed with dsbA/dsbC
Endoxylanase Bacillus sp. Alkaline phosphatase E. coli 5.2 g/L in periplasm under high cell Choi et al. (2000)
(AP) density cultivation

(Watanabe et al. 2009). Among those peptides, 11 signal
peptides conferred a 50- to 150-fold higher secretion level
than the well-known corynebacterial secretory protein PS2.
As the authors did not test the best signal peptides using
another heterologous protein, this improvement in perfor-
mance may not be applicable for other proteins.
Another approach utilises the proteomic profiles, partic-
ularly the secretome, of a host cell to identify better-
performing signal peptides. The rationale of this strategy
assumes that abundantly secreted proteins should have an
above-average signal peptide efficiency, making these sig-
nal peptides ideal for high levels of recombinant protein
secretion. Two-dimensional gel electrophoresis followed
by mass spectrometry is used to determine the identity of
secreted proteins and then its associated signal peptide. In
addition to being a tool to verify genome-based signal
peptide predictions, this approach avoids the tedious and
laborious tasks of constructing a signal peptide library and

Table 2 Several in silico tools for signal sequence prediction

Prediction tools Prediction methods URL References

SignalP Hidden Markov model and neural network cbs.dtu.dk/services/SignalP/ Petersen et al. (2011)
PrediSi Position weight matrix www.predisi.de/index.html Hiller et al. (2004)
Signal-BLAST Sequence alignment (BLASTP) sigpep.services.came.sbg.ac.at/signalblast.html Frank and Sippl (2008)
Signal-3L Multi-program modules www.csbio.sjtu.edu.cn/bioinf/Signal-3L/ Shen and Chou (2007)
Signal-CF Multi-program modules www.csbio.sjtu.edu.cn/bioinf/Signal-CF/ Chou and Shen (2007)
Phobius Hidden Markov model phobius.sbc.su.se/index.html Kall et al. (2007)
SIG-Pred Matrix bmbpcu36.leeds.ac.uk/prot_analysis/Signal.html –
SPEPLip Neural Network gpcr.biocomp.unibo.it/cgi/predictors/spep/pred_ Fariselli et al. (2003)
spepcgi.cgi
OCTOPUS BLAST homology and neural network octopus.cbr.su.se/index.php Viklund and Elofsson
and hidden Markov model (2008)
3816
evaluating secretion efficiencies. For diderm organisms,
such as E. coli, one could perform proteomic verification
using the periplasmic or extracellular fraction. While both
fractions serve well to identify native signal peptides, pro-
teomic analysis of the extracellular fraction may benefit
those who favour excretory production of recombinant pro-
teins. In general, the N-terminal signal sequence together
with its mature protein is needed for excretion of recombi-
nant protein, but the mechanism for passing through the
outer membrane is largely unknown. With this approach,
Qian et al. (2008) isolated 22 proteins that were abundant in
the fermentation medium of E. coli BL21(DE3). Among those
potential candidates, OsmY (a periplasmic, osmotically induc-
ible protein Y) was found to be an efficient fusion carrier to
direct excretion of recombinant proteins (E. coli alkaline
phosphatase, human leptin and B. subtilis α-amylase), with
concentrations ranging from 250 to 700 mg/L under high cell
density cultivation conditions. The fusion of recombinant pro-
teins to different segments of OsmY yielded localisation to
different cell compartments, namely, the cytoplasm for the
protein fused to the mature osmY, the periplasm for the protein
fused to the signal sequence of osmY and the extracellular
medium for the protein fused to the full-length osmY (signal
sequence and mature osmY). The usefulness of OsmY was
also shown in recent work by Kotzsch et al. (2011). The
excretory production of 24 human target proteins was higher
when the proteins were fused to OsmY instead of with OmpA
or OmpF. A protease cleavage site (TEV site) and 6xHis tag
was located between the fusion carrier and the heterologous
protein to facilitate the removal of fusion carrier and rapid
purification, respectively. In this case, highly purified protein
samples (in the range of 100–2,700 μg from a 750-ml culture)
were obtained from different groups of human target proteins
when fused to OsmY. Importantly, the authors suggested that
OsmY may facilitate the solubility, stability and folding of
target protein. These studies noted the importance of the
proteomic approach to isolating novel signal peptides and
excretion factors for high-level secretion of recombinant pro-
teins. Another version of the proteome-based method identi-
fied potential signal peptides using whole-colony matrix-
assisted laser desorption–ionization time-of-flight mass spec-
trometry (MALDI-TOF MS). This high-throughput method
identifies extracellular proteins directly from intact bacterial
colonies and has been used for the identification of type VII
secretory proteins from Mycobacterium marinum and type III
secretion systems from Pseudomonas aeruginosa (Champion
et al. 2012). This method specifically detects extracellular
proteins that attach to the cell surface, limiting its detection
to a subset of protein secretion systems.
The approaches outlined previously require a heavy in-
vestment in labour (signal peptide library construction and
high-throughput screening) and/or advanced equipment
(two-dimensional gel electrophoresis and MALDI-TOF
Appl Microbiol Biotechnol (2013) 97:3811–3826
MS), which restricts the use of these approaches in labora-
tories with limited resources. An additional approach that
may ameliorate these difficulties involves the ‘rational
screening’ of available signal sequences from the literature
and from protein databases. The availability of public data-
bases of signal sequences has increased the interest in this
method as an easy and less resource-heavy solution. Table 3
lists several signal sequence databases that are accessible
from the Internet. Among those, CoBaltDB (Goudenege et
al. 2010) represents the most comprehensive and systematic
database for predicting protein localisation in bacterial and
archaeal microbes, integrating 43 in silico localisation tools
for the proteome of 784 organisms and displaying relevant
information from NCBI and Kyoto Encyclopedia of Genes
and Genomes (KEGG) for a given protein. An easy and
rapid analysis of the proteome of an organism can be made
with a high degree of confidence. Signal sequences that
consistently score as a potential signal peptide across a
range of prediction tools can be selected for further analyses,
effectively narrowing the search space and increasing the
possibility of detecting an efficient signal peptide. As each
database has its own strengths and weaknesses, the choice
will depend on the purpose of study.
Signal peptide modification
Protein secretion largely depends on the efficiency of the
signal peptide in driving protein translocation. Minor mod-
ifications to the natural signal peptide can often significantly
improve the secretory expression over that of the parent
peptide. In this section, we discuss several important con-
siderations for the design of an improved signal peptide for
the expression in E. coli.
The n-region: position of the basic residues
The n-region, as its name implies, is located at the extreme
N-terminal of a Sec signal peptide. A key characteristic of
this region is the net positive charge, which arises from one
or more basic residues. The positive charges (or basic resi-
dues) play several roles in the protein translocation process,
enabling the signal peptide to insert itself into the inner
membrane by forming a loop with the negatively
charged lipid head groups via electrostatic interactions
(Inouye and Halegoua 1980). The highly basic n-region
may also promote interactions with SRP, influencing the
signal peptide’s route to the SecA/SecYEG translocase
(Peterson et al. 2003). Removing the basic residues (by
reducing the length or substituting with neutral/acidic
residues) has various effects on the export process,
including a reduced rate of export (Nesmeyanova et al.
1997), a decreased rate of protein synthesis (Inouye et
al. 1982), a change in the signal peptide cleavage site
Appl Microbiol Biotechnol (2013) 97:3811–3826 3817

Table 3 List of several publicly available signal sequence databases

Database Target Features URL Reference

Effective Bacterial signal Predicts effector (proteins with a effectors.org Jehl et al. (2011)
sequences signal peptide and eukaryotic
domain) in bacterial genome
(symbiotic and pathogenic)
SPdb Signal sequences Experimentally verified signal proline.bic.nus.edu.sg/spdb/ Choo et al. (2005)
of archaea, sequence from Swiss-Prot
prokaryotes and
eukaryotes
CoBaltDB Bacterial and archaeal Integrates 43 in silico tools to www.umr6026.univ-rennes1.fr/english/ Goudenege et al.
proteome predict protein localisation home/research/basic/software/cobalten (2010)
from genome sequence;
provides links to external
databases (NCBI and KEGG)
Signal Peptide Signal peptides from Collection of signal peptides www.signalpeptide.de/ –
Website eukaryotes, from UniProt Knowledgebase
prokaryotes Release 14.7
and viruses
EXProt Gram-positive and Uses a weight matrix algorithm 142.51.14.12/Laurentian/Home/Departments/ Saleh et al. (2001)
gram-negative and two artificial neural Biology/Faculty_and_Staff/Professors/
signal peptides networks to predict signal saleh/
peptides from genome sequence ExProt.htm?Laurentian_Lang=en-CA
Swiss-Prot Prokaryote (426) and Experimentally verified signal ftp.ebi.ac.uk/pub/contrib/swissprot/testsets/ Menne et al. (2000)
signal test eukaryote (1,142) peptide dataset signal/data/
set signal peptides

(Suominen et al. 1995) or no discernible effects (Vlasuk
et al. 1983).
As these substitution experiments suggest that the pres-
ence of (one or more) basic residues in the n-region may be
necessary for the development of a good signal peptide,
optimisation of the peptide may require an examination of
the presence and location of these residues. For maximal
secretion performance, two factors need to be taken into
consideration: (1) the positions of the basic residues and
(2) the magnitude of the net positive charge. The presence of
a basic residue immediately after the start codon (the second
codon, P2) appears to improve the rate of protein secretion.
For instance, the signal peptide of MBP and ribose-binding
protein had high export rates when basic residues (i.e. lysine
and arginine) were located at P2 (Puziss et al. 1992).
Similarly, signal peptide variants of alkaline phosphatase
harbouring lysine, histidine or tyrosine at P2 yielded large
quantities of exported mature alkaline phosphatase
(Nesmeyanova et al. 1997). Basic residues, particularly
lysine, at P2 induced the accumulation of anionic phospho-
lipid, most likely through an increased interaction between
the signal peptide and the inner membrane. Anionic phos-
pholipids are essential for efficient protein transport both in
vivo (de Vrije et al. 1988) and in vitro (Phoenix et al. 1993)
and have been shown to promote the interaction of a pre-
cursor protein with phospholipids in a model membrane
system (Keller et al. 1996). Signal peptides may establish
ionic pairs with the negative charge of the phosphate groups
of the lipid molecule (Nesmeyanova et al. 1997) and the
acidic residues of SecA (Gelis et al. 2007), thus stabilising
the hydrogen bonding of the signal peptide α-helix
complex.
The preference for a basic residue, particularly lysine, at
P2 is challenged by recent findings from Zalucki et al.
(2007), which argued with experimental evidence that a
strong initiation of translation—and not the positive
charge—at P2 confers a more efficient signal peptide.
Coincidentally, the best initiator codon is AAA (which
codes for lysine), a basic residue that is commonly found
at P2 of E. coli natural signal peptides. The codon AAT
(which codes for asparagine; neutral) is the second best
initiator codon after AAA. Substitution of AAA with AAT
at P2 of the MBP and alkaline phosphatase signal se-
quences did not significantly change the exported levels
of β-lactamase. In contrast, substitution of AAA with a
weak initiator codon at P2 of the MBP signal peptide
resulted in a significant decrease in protein export. In an-
other example, a fusion with an N-terminal signal sequence
(PelB, OmpA and CSP)—containing the AAA codon at
P2—led to a strong stimulation of expression of three
difficult-to-express proteins (granulocyte–macrophage
colony-stimulating factor (GM-CSF), interferon alpha 2b
and single-chain antibody variable fragment) (Sletta et al.
2007). The increased transcription and translation levels
arising from the more stable mRNA species were suggested
to be the primary factor behind the improved expression.
3818
The presence of a basic residue or AAA codon at P2 does
not always guarantee an optimum recombinant protein se-
cretion. Through saturation mutagenesis of the positively
charged n-region (positions 2–7) of a B. subtilis α-amylase
signal peptide, Caspers et al. (2010) isolated four single-
point mutations that resulted in increased quantities of
cutinase (onefold to twofold increase compared with the
wild type). Three of these mutations reduced the net charge
from +3 to +2 (F2D, F2E, K4L and F6W). The mutations
did result in a slow rate of recombinant protein synthesis,
thus preventing a jamming of the secretion translocase and
the induction of cell-associated proteases and stress-related
genes, in addition to enabling high levels of protein secre-
tion and processing.
Current evidence suggests that a functional n-region
should possess a net charge of at least +1 for the efficient
export of the recombinant protein and that different signal
peptides may require different magnitudes of positive
charge for maximum efficiency. For example, export of the
MBP was the most favourable for signal peptides with at
least a net charge of +1 (Puziss et al. 1992). The maturation
of PhoA was also most efficient in the presence of a +1 net
charge (lysine, histidine or tyrosine) (Nesmeyanova et al.
1997). In another example, a mutant L-asparaginase II signal
peptide with a +5 net charge had higher intracellular solu-
bility and periplasmic export of a recombinant cyclodextrin
glucanotransferase in E. coli compared with the parent sig-
nal peptide (net charge of +2) (Ismail et al. 2011).
The h-region: levelling off of hydrophobicity
The hydrophobic core, generally known as the h-region, is a
hallmark feature of signal peptides, functioning like a secu-
rity pass for the secreted protein to exit through the guarded
gate—SecA/SecYEG. Two characteristics—helical propen-
sity and hydrophobicity level—are important for the recog-
nition of the signal peptide by SecA. Upon contact with the
SecA hydrophobic groove (the substrate binding site), the
highly hydrophobic signal peptide shifts into a helical con-
formation (Chou and Gierasch 2005), triggering the SecA-
ATPase activity (Kebir and Kendall 2002). Either a deletion
or a substitution of the hydrophobic residues dramatically
impairs protein export (Suominen et al. 1995; Izard and
Kendall 1994), resulting in pre-protein accumulation at the
cell membrane with a non-native conformation. A sufficient
level of hydrophobicity is needed for efficient processing of
the signal peptide and translocation of the protein. Rusch et
al. (1994) demonstrated that at least five leucine residues
were required in the h-region for the efficient function of an
alkaline phosphatase signal peptide.
Increasing the hydrophobicity levels of the h-region can
improve the rate of protein secretion. As Chen et al. (1996)
elegantly demonstrated using a signal peptide competition
Appl Microbiol Biotechnol (2013) 97:3811–3826
assay, the hydrophobicity levels of the signal peptide deter-
mined its affinity towards the protein secretion pathway in
E. coli. The assay involved the expression of a modified
phoA that possessed two signal peptides arranged in tandem
(signal peptide 1–signal peptide 2–PhoA). The efficiency of
the signal peptide was determined from the preference of the
signal peptide cleavage point (utilisation of the first signal
peptide (signal peptide 1) was indicated by the signal pep-
tide cleavage at the first site and vice versa). Mutant signal
peptides with a marginal increase in hydrophobicity and
length (by addition of leucine in the h-region) effectively
outcompeted the original signal peptide for secretion effi-
ciency (Chen et al. 1996). Signal peptide variants with up to
ten leucine residues in the hydrophobic core were efficiently
translocated and cleaved. The authors suggested that an effi-
cient signal peptide should have a high ‘hydrophobic density’
in the core region. This hydrophobic core promotes high
levels of protein secretion, with a highly hydrophobic h-
region compensating for a ‘weaker’ or suboptimal n-region.
For instance, the translocation of E. coli lipoprotein was
insensitive to the n-region positive charge for OmpF signal
peptides containing nine or more leucine residues in the
hydrophobic core (Hikita and Mizushima 1992b). At a low
hydrophobicity level (eight or fewer leucines in the h-region),
protein translocation was dependent on the n-region positive
charge, with the best signal peptide containing four lysine and
eight leucine residues (MMKKKK LLLLLLLL GTANAAS).
A subsequent in vitro experiment (exerted membrane vesicles
of E. coli) revealed that the total hydrophobicity and the length
played a role in determining the secretion efficiency (Hikita
and Mizushima 1992a). OmpF signal peptides with a stretch
of eight leucine residues (MMKRN LLLLLLLL GTANFPG)
or ten alanine–leucine residues (MMKRN • ALALALA
LA• GTANFPG) in the hydrophobic core translocated large
quantities of lipoprotein. Similarly, a site-directed mutagene-
sis coupled with a pulse-chase analysis demonstrated that an
h-region with five or more leucine residues rendered the n-
region net charge unimportant for determining protein secre-
tion levels (Rusch et al. 2002). These findings are in agree-
ment with early cross-linking data that suggested that the
interactions between the signal peptides and SecA are propor-
tional to the length (Mori et al. 1997) and hydrophobicity
levels of the h-region (Valent et al. 1998).
The extent of the effect of h-region hydrophobicity on the
optimisation of the signal peptide may depend on the pas-
senger protein. As stated earlier, highly hydrophobic signal
p e p ti d e s p r o m o t e S R P - d e p e n d e n t r o u t e s t o t h e
SecA/SecYEG translocon. Increasing the hydrophobicity
of natural signal peptides may switch the targeting route
from a SecB-dependent to an SRP-dependent pathway. For
example, a LamB signal peptide with four additional hydro-
phobic residues avoided recognition by SecB and was
rerouted to an SRP-dependent pathway for export, differing
Appl Microbiol Biotechnol (2013) 97:3811–3826
from its less hydrophobic parent peptide (Bowers et al.
2003). In this case, the extent of the SecB and SRP pathway
efficiencies for recombinant protein secretion was not deter-
mined. A secreted protein that requires some sort of post-
translational modification might benefit from the SecB path-
way (Bensing et al. 2007), as the SRP-dependent pathway
occurs co-translationally, preventing any form of cytoplas-
mic modification. By using SRP-dependent signal peptides
(i.e. DsbA, TorT, TolB or SfmC), Steiner et al. (2006)
produced more than a 1,000-fold enrichment of phage-
displayed protein compared with the Sec-dependent signal
peptides (i.e. LamB, MalE, MglB, OmpA, PelB or PhoA).
The unnecessary processing of the phage-displayed protein
that follows the SecB route stalled in the cytoplasm and
retarded the secretion of the protein. Non-problematic pro-
teins may benefit from exportation through the SecB-
dependent pathway, as the SecB pathway approach has a
large capacity for protein export in E. coli (more than 90 %
of secreted protein in E. coli follows a SecB-dependent
route). As stated earlier in this article, SRP binds to highly
hydrophobic signal peptides that form long α-helix struc-
tures. To reduce the affinity of highly hydrophobic signal
peptides towards SRP, a helix breaker (particularly glycine)
can be introduced within the hydrophobic core of the signal
peptide. Extending the α-helix structure, through substitu-
tion of glycine with cysteine and leucine (mutations G10C
and G10L), of a PhoE signal peptide significantly shifted the
affinity of this peptide towards the SRP component (P48)
and away from the SecB pathway (Adams et al. 2002).
Similarly, the replacement of glycine within the hydropho-
bic region of the GspB signal peptide reduced the accessory
Sec-dependent transport (i.e. SecB), inducing canonical
Sec-dependent exportation (Bensing et al. 2007). The pres-
ence of a helix breaker in the hydrophobic core of the signal
peptide can dramatically affect recombinant protein export.
The introduction of a glycine residue in the middle of the h-
region doubled the periplasmic and extracellular recombi-
nant cyclodextrin glucanotransferase activity, compared
with the no-glycine, wild-type signal peptide (Jonet et al.
2012; Low et al. 2012). The position of the helix breaker
(glycine) played a role in protein export levels. A glycine
residue at position −7 improved the pre-protein export com-
pared with a glycine residue at position −5 or −9 (the
predicted total length of the h-region was 12 residues).
This increase in the export phenotype may have resulted
from a change of the targeting pathway or another
unidentified mechanism.
Reports in the literature have suggested that the best signal
peptide should be the most hydrophobic and have a glycine
residue in the middle of the hydrophobic core. At first glance,
this may seem logical to some extent, but there are several
difficulties that prevent this sequence from becoming a work-
able solution. As demonstrated by Rusch et al. (1994), highly
3819
competitive signal peptides (with a ten-leucine h-region) sig-
nificantly inhibited the translocation of native β-lactamase.
Reduced expression/processing of the native protein may
have severely influenced important cell functions and retarded
cell growth, creating a major problem in the up-scaling to
production. In this case, an optimisation of the hydrophobicity
levels by substituting less hydrophobic residues with more
hydrophobic residues (e.g. leucine) should be performed, one
residue at a time, until an optimal balance between protein
secretion and cell growth is obtained.
The c-region: beyond the ‘−3, −1 rule’
After passing through the SecYEG translocon, the signal
peptide is embedded into the inner membrane and has to be
cleaved off from the mature protein to complete the secre-
tion process. The cleavage of the signal peptide requires the
recognition and proteolytic action of signal peptidase at a
specific site on the signal peptide. This site is commonly
known as the c-region, and it is most likely the least ambig-
uous region to optimise. Based on the involvement of the c-
region with signal peptidase I in a specific enzyme–substrate
interaction, special limits on the sequence characteristics,
conformation and polarity may be imposed on the cleavage
site in the c-region. The most prominent rule is the ‘−3, −1
rule’ or AXA motif (von Heijne 1984), with position −1 and
−3 preceding the cleavage site favouring residues with small
neutral side chains (particularly alanine). The c-region
should have a five-residue length to ensure both high export
and cleavage efficiency (Suominen et al. 1995). The effi-
ciency of the signal peptide cleavage significantly influ-
ences protein secretion levels. Using a novel biosensor
technique, Geukens et al. (2004) demonstrated that the
cleavage of the signal peptide was the rate-limiting step
for protein secretion in Streptomyces lividans. The authors
also reported that the signal peptidase interacted with the
signal peptide at the region −8 to +1. In another example,
coexpression of signal peptidase I (sipM) increased
dextransucrase secretion in Bacillus megaterium by 3.7-fold
(Malten et al. 2005).
Recent studies have further detailed the requirements for
an efficient c-region. Using a directed evolution approach,
Karamyshev et al. (1998) provided new experimental evi-
dence to support the ‘−3, −1 rule’, with alanine preferred for
position −1, while position −3 can accommodate several
residues (i.e. alanine, serine, glycine, leucine and cysteine).
Position −2 favoured large amino acids (i.e. phenylalanine,
tyrosine, leucine and histidine) for efficient processing. This
observation was similar to the report by Wrede et al. (1998)
on the design of the c-region using computer-based tech-
niques. Position −4 favoured serine, phenylalanine, tyrosine,
leucine and cysteine, but not lysine and glycine. Position −5
preferred mid-sized amino acids, such as serine. A
3820
conformational analysis of the cleavage site (−5 to −1 region)
suggested that an extended β-conformation was optimal for
the signal peptidase binding pocket/active site. This finding is
in good agreement with the available X-ray structure of E. coli
signal peptidase (Paetzel et al. 1998). By using in silico
docking, the authors showed that the side chain of alanine at
position −1 interacted with the signal peptidase pocket S1
(formed by methionine-91, isoleucine-144, leucine-95 and
isoleucine-86). The side chain of alanine at position −3 ac-
commodated a shallow hydrophobic pocket S3 (formed by
phenylalanine-84, isoleucine-86, isoleucine-101, valine-132,
isoleucine-144 and aspartic acid-142). The S3 pocket was
larger than the S1 pocket, making room for a larger amino
acid (i.e. valine, leucine and isoleucine) at the −3 position. The
stringent substrate specificity of the signal peptidase contrib-
uted to the orientation of the signal peptide’s side chain
towards the binding site and to the hydrogen bonds between
the signal peptidase and the signal peptide at positions −3, −2
and −1 (Choo et al. 2005).
Another approach to optimise the c-region sequence is de
novo design using computer-based techniques. The combina-
tion of an artificial neural network and an evolutionary dis-
tance matrix is an attractive method for designing a functional
and efficient c-region. For instance, Paul Wrede’s group
designed an ‘idealised’ cleavage site using the (1, λ)-evolution
strategy and artificial neutral networks to generate optimum c-
region sequences and to estimate the quality of the generated
sequences, respectively (Schneider and Wrede 1994; Wrede et
al. 1998). They demonstrated that the Miyata matrix (Miyata
et al. 1979), which serves as a calculator for the evolution
distance measurement between amino acids, produced higher-
quality sequences. In addition to confirming the ‘−3, −1 rule’,
the authors also indicated the amino acid preferences at posi-
tions −6, −2 and −7 to −10, with alanine at position −6, a
relatively large residue (such as tryptophan) at position −2 and
phenylalanine (hydrophobic residue) at positions −7 to −10.
The predicted ‘idealised’ c-region (SME2) was reported to be
FFFFGWYGWA↓AC, where ‘↓’ indicates the cleavage point.
SME2 fused to a ribonuclease T1 was recognised and cleaved
by signal peptidase I as efficiently as OmpA signal peptide
(LAGFATVAQA↓AC) (Wrede et al. 1998), and the exported
quantity of ribonuclease protein, measured using SDS-PAGE,
appeared to be higher relative to that obtained with the OmpA
signal peptide.
The pro-region: the fourth musketeer (factor) in action?
The presence of a secretion signal does not always guarantee
an efficient secretion of the recombinant protein.
Problematic pre-proteins that fold prematurely or aggregate
can be translocated by using an overexpression of chaper-
ones to reduce their folding rate (Park et al. 2004; Kwon et
al. 2002). Another important region to consider is the pro-
Appl Microbiol Biotechnol (2013) 97:3811–3826
region, also termed the ‘export initiation domain’, which
extends up to 30 residues long immediately downstream of
the signal peptidase cleavage site (Andersson and von
Heijne 1991). The nature of the charged residues in this
region influences the discharge of the pre-protein across the
inner membrane of E. coli. This region is largely hydrophil-
ic, and deletions in this region can cause proteins to accu-
mulate in the cytoplasm and aggregate as inclusion bodies
(Kim et al. 2007). An introduction of basic residues into this
region can block (Andersson and von Heijne 1991; Geller et
al. 1993) or reduce protein export (Campion et al. 1997) and
change the signal peptidase I cleavage point (Itoh et al.
1990), regardless of the targeting pathway (SecB-dependent
or SRP-dependent) (Tian and Bernstein 2009). Furthermore,
a recombinant protein fused at position +2 (relative to the
signal peptide cleavage point) of a signal peptide was more
efficiently secreted than the protein fused at position +1
(Geukens et al. 2004), suggesting that the pro-region (posi-
tion +1) is involved in protein translocation. Experiments
using in silico docking suggested that the signal peptidase
interacts with the signal peptide not only at the AXA site but
also in the pro-region, from position +1 to +6 (Choo et al.
2005). A statistical analysis of 107 experimentally deter-
mined signal peptidase substrates suggested a preference for
certain residues in this region (position +1 to +6). The
validity of these findings requires further analysis.
In contrast with the n-region, the pro-region requires a net
negative charge for efficient protein export. For example,
substitution of the basic residues with acidic residues dras-
tically increased recombinant protein export (Campion et al.
1997; Yamanaka and Okamoto 1996). The need for a neg-
atively charged pro-region is not unique to E. coli; this
requirement was also observed in other gram-positive bac-
teria (Kajikawa et al. 2010; Kouwen et al. 2010), suggesting
that the pro-region is actively involved in both pre-protein
targeting and translocation. The optimal values of both the
magnitude of the charge and the position of the acidic
residues may depend on the target protein. Recombinant
phosphoglycerate kinase required a net charge of −2 or −3
on the first 15 residues for efficient export, while the en-
zymes glyceraldehyde-3-phosphate dehydrogenase and eno-
lase required a net charge of −6 (Tian and Bernstein 2009).
The net negatively charged pro-region might form loop struc-
tures with the positively charged n-region, complementing the
membrane electrochemical potential (the ‘inside positive
rule’). The membrane potential across the E. coli inner mem-
brane is characterised by a positive charge on the periplasmic
surface. Kaderbhai et al. (2004) classified the performance of
protein exportation based on the nature of the two residues
immediately adjacent to the signal peptide cleavage site (po-
sition +1 and +2). Hyperexporters contained acidic residues,
hypoexporters contained basic residues or cysteine and inter-
mediate exporters containing predominantly neutral residues.
Appl Microbiol Biotechnol (2013) 97:3811–3826
Hyperexporters bound less frequently to the membrane com-
pared with hypoexporters and intermediate exporters. Indeed,
a comparative analysis of the E. coli and B. subtilis cytoplas-
mic and secretory proteins showed that most secretory pro-
teins have neutral or net negative charges for the first 10–15
residues (Tian and Bernstein 2009).
The steric bulkiness of amino acids at position +1 is
another factor that may influence protein export. Smaller
residues, such as cysteine, glutamine, threonine and serine,
at position +1 ensured high export levels of the mammalian
globular cytochrome b5 precursor in E. coli (Kaderbhai et al.
2010). The authors suggested that large (i.e. isoleucine) and
rigid (i.e. proline) amino acids at position +1 may restrict the
activity of signal peptidase I on the signal peptide (steric
hindrance), causing a lower cleavage and discharge level of
the mature protein. Small and flexible amino acids in the
pro-region allowed a rapid maturation with improved export
levels of subtilase, possibly due to the tendency of the
secreted protein to maintain a translocation-competent state
(Fang et al. 2010). In another example, a deletion of the pro-
region (n-terminal 16 residues) rendered transglutaminase
secretion-defective and insoluble (Liu et al. 2011). A model
of the protein suggested that the pro-region may be involved
in protein folding, suggesting that this region is essential for
efficient protein secretion.
Signal sequence as an intramolecular chaperone
The role of the signal peptide in protein secretion is not
limited to the interactions with the secretory machinery (i.e.
SecA, signal peptidase), but also onto the passenger protein.
Signal peptides can reduce folding rates (Tomkiewicz et al.
2008) and change both the kinetic and thermodynamic
destabilisation of the passenger protein (Beena et al. 2004),
independent of the folding nature of the target protein.
Together with SecB, the signal peptides (particularly the hy-
drophobic core of the peptide) can interact with the hydropho-
bic regions of the passenger protein to maintain a non-native
conformation and a transport-competent state—properties that
are crucial for all secreted proteins. Recent findings suggest
that signal peptides can be engineered to not only enhance
protein export but also to improve the physiological state of
the cell. By increasing the net charge on the n-region of the L-
asparaginase II signal peptide (from +2 to +5), Ismail et al.
(2011) increased both the protein solubility and the periplas-
mic export of recombinant cyclodextrin glucanotransferase in
E. coli compared with the wild-type signal peptide. Cell
viability also improved significantly, most likely due to a
strong cell membrane. More studies should be performed to
confirm these observations. One possible explanation may be
that the mutant signal peptide may relieve a phenomenon
called ‘saturated translocation’ (Fu et al. 2005), which is
caused by an accumulation of the transport-deficient pre-
3821
protein at the SecYEG translocon, preventing the secretion
of the endogenous proteins that are important to maintain cell
survival.
Conclusions and future prospects
Constructing a system of secretory expression for recombi-
nant proteins is a simple task, but achieving a substantially
high level of production requires one to balance between
gene expression, the protein transport process, cell fitness
(viability) and the composition of the medium. For the
process of protein transport specifically, the main obstacles
can be ameliorated with an improved understanding of the
mechanisms of protein secretion. The Sec signal peptide
may appear to be a simple and redundant molecule, but this
short polypeptide contains all of the information needed to
drive protein secretion through a sequential interaction with
the Sec machinery and the passenger protein. Recent find-
ings have suggested that the distinct regions of the signal
peptide (i.e. the n-, h-, c- and pro-regions) do not work
independently but are organised and function in a coopera-
tive manner. A summary of a more efficient signal peptide
design is proposed as in Fig. 2. We suggest that an ideal
signal peptide should have an optimal and balanced ‘design’
of properties for each of the distinct regions. For this ex-
pression system, solving the X-ray structure of the Sec
machinery (i.e. SecA and the signal peptidase) could pro-
vide details as to their interactions with the signal peptide.
The future of recombinant protein production may rely
heavily on using modified microbial cell factories with an
efficient protein secretion (and/or excretion) system. The
search for a more efficient signal peptide and secretion
pathway will remain as an important highlight and challenge
to realising that goal. Several exciting challenges that re-
main are described as follows:
1. The hydrophobic core is most likely the most dominant
characteristic in influencing the capacity and efficiency
of a signal peptide during protein secretion. Despite its
importance, little systematic work has been performed
on engineering an improved hydrophobic core. Current
research has suggested that an enhancement in the h-
region’s efficiency may require an increase in hydro-
phobicity levels. Less is known about the effect of
different hydrophobic residues in the hydrophobic core
on the efficiency of the signal peptide in driving trans-
location. Two questions that remain to be answered
include the following: Are highly hydrophobic residues
(i.e. isoleucine) favourable over less hydrophobic or
non-hydrophobic residues (i.e. alanine, glycine) or vice
versa? Is there a maximum hydrophobicity threshold for
better signal peptide design? The recent discovery that
3822
glycine (a non-hydrophobic residue) and its location in
the h-region significantly influenced the secretion of
recombinant proteins in E. coli (Jonet et al. 2012) re-
vealed our limited understanding of this region. An
exhaustive approach, such as directed evolution, that
probes the effects of different residues in the hydropho-
bic core could provide an improved understanding of
the fundamental requirements for the hydrophobic core.
2. One of the more ambitious goals in signal peptide
research is the development of a cross-host, universal
secretion signal peptide. The ability of signal peptides to
be recognised and translocated in a wide range of hosts
would ensure flexibility in protein production, simpli-
fying the optimisation processes. Early studies have
demonstrated that several natural signal peptides exhibit
some degree of functionality in other heterologous ex-
pression hosts (Uhlen and Abrahmsen 1989; Allet et al.
1997; Golden et al. 1998; Coloma et al. 1992;
Kirkpatrick et al. 1995; Gray et al. 1985; Asakura et
al. 1995). Humphreys et al. (2000) demonstrated that
eukaryotic signal sequences can be engineered to per-
form more efficiently in E. coli through codon optimi-
sation at the 5′ end of the coding sequence. Wang et al.
(2001) and Tan et al. (2002) also engineered a novel
secretion signal peptide that was capable of targeting
recombinant protein secretion in E. coli, Saccharomyces
cerevisiae and six different eukaryotic cell lines. The
authors outlined several key characteristics for cross-
host secretion signal peptides, including the ability to
contain the three typical domains observed in all signal
peptides, to obey the ‘−3, −1’ rule and to comprise an
optimal ratio of charge-to-hydrophobicity level (Tan et
al. 2002). Enhancing the performance of the secretion
signal in multiple expression hosts while maintaining
flexibility (i.e. functionality in multiple host cells) can
be challenging. The use of computational design to
match and complement the requirements of efficient
secretion in multiple expression hosts should produce
desirable outcomes. Genome-scale comparisons of sig-
nal peptides between common prokaryotic hosts (i.e. E.
coli and B. subtilis) and eukaryotic hosts (i.e. CHO, S.
cerevisiae) could reveal similarities and differences in
signal peptide architecture and design. Furthermore, the
recent discovery of the X-ray structure of Sec machin-
ery (i.e. SecA) may also aid in the understanding of
signal peptide interactions with the respective systems.
3. Protein expression and secretion in biological systems are
complex processes that involve a plethora of sequential
protein–protein interactions. Improving recombinant pro-
tein secretion should not be confined solely to engineering
more efficient interactions between the signal peptide and
the Sec machinery. Systems biotechnology, a systems-
level approach that considers all biological processes in
Appl Microbiol Biotechnol (2013) 97:3811–3826
a living organism for the generation of knowledge and
products, can be used to achieve the best performance in
an integrated manner. In this case, E. coli is advantageous,
as it is the most well-known organism with the most
complete genome-scale metabolic reconstruction model
(Orth et al. 2011). A recent study demonstrated the feasi-
bility and practicality of this approach. By using a 13C
metabolic flux analysis, Umakoshi et al. (2011) identified
the rate-limiting step (NADH/NAD + balance) in
transglutaminase secretion in C. glutamicum,
counteracting the negative effects and generating a 1.4-
fold increase in the production of the target protein.
4. Another area that is worth pursuing is the potential
activity of the signal peptide as a molecular chaperone.
Recent evidence suggests that the ‘influence’ of signal
peptide is exerted not only onto its passenger protein but
may extend towards the well-being of host cell (cell
physiology, cell viability, etc.). A mutant signal peptide
with an increased positive charge in the n-region (+5)
significantly improved both the cell viability and the
recombinant protein secretion compared to the wild-
type signal peptide (+2) (Ismail et al. 2011). Whether
this phenomenon is the consequence of improved pro-
tein secretion (i.e. reduced inclusion body formation,
reduced stress, de-saturation of protein translocon,
etc.) needs further clarification. The question remains
of how the signal peptide reacts with cellular machinery
to perform such functions. One may rely on advanced
computational algorithms (i.e. molecular dynamics
analysis) to simulate protein folding and reveal possible
interactions between the signal peptide and its passen-
ger protein. A systems-level ‘omics’ approach, such as
transcriptomics or proteomics, could provide insight on
the effect of the design of the signal peptide on the cell.
Acknowledgements We would like to thank Noor Faizah Ismail for
the critical reading of the manuscript. Support from Abdul Munir
Abdul Murad, Amir Rabu and Farah Diba Abu Bakar of Universiti
Kebangsaan Malaysia and Raha Abdul Rahim of Universiti Putra
Malaysia is greatly appreciated. This work was supported by the
Genomics and Molecular Biology Initiatives Programme of the Ma-
laysia Genome Institute, Ministry of Science, Technology and Innova-
tion Malaysia (Project No. 07-05-MGI-GMB011) and the Exploration
Research Grant Scheme (R.J130000.7835.4L046), Universiti
Teknologi Malaysia. Kheng Oon Low is a researcher of Universiti
Teknologi Malaysia under the Post-Doctoral Fellowship Scheme.
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