Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli

Article abstract of DOI:10.1016/j.peptides.2010.08.008

Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for large-scale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10 L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.

Full text of DOI:10.1016/j.peptides.2010.08.008

Peptides 31 (2010) 1957–1965
Contents lists available at ScienceDirect
Peptides
journal homepage: www.elsevier.com/locate/peptides
Cost-effective expression and purification of antimicrobial and host defense
peptides in Escherichia coli
B. Bommariusa, H. Jenssenb,c, M. Elliottb, J. Kindrachukb, Mukesh Pasupuletib, H. Gierend, K.-E. Jaegerd,
R.E.W. Hancockb, D. Kalmana,∗
a Emory University, Atlanta, GA, USA
b University of British Columbia, BC, Canada
c Roskilde University, Denmark
d Institute for Molecular and Enzyme Technology, University of Düsseldorf, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 June 2010
Received in revised form 2 August 2010
Accepted 3 August 2010
Available online 14 August 2010
Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of
microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-
resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic
application. High manufacturing costs associated with amino acid precursors have limited the delivery
of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of
peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity
of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of
recombinant peptides often requires multiple steps and has not been cost-effective. Here we have devel-
oped methodologies appropriate for large-scale industrial production of HDPs; in particular, we describe
(i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria
without significant toxicity and (ii) a simplified 2-step purification method appropriate for industrial
use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5
and E6). Using this technology, pilot-scale fermentation (10 L) was performed to produce large quantities
of biologically active cationic peptides. Together, these data indicate that this new method represents
a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good
Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.
© 2010 Elsevier Inc. All rights reserved.
Keywords:
Antimicrobial peptide
Host defense peptide
Bacterial expression system
1. Introduction
(HDPs) [13]. These are characterized by being short (12–50 amino
acids), having an overall positive charge of +2 to +9, and sufficient
The increase in resistance of bacterial strains to existing antibi-
otics is a major public health concern (WHO (2008) World Health
Statistics 2008. World Health Organization Press), and has spurred
intensive efforts to develop new classes of antibiotics that are
effective against antibiotic-resistant strains, with limited success
[37]. The anti-infective properties of HDPs, evolutionary conserved
biodefense molecules found in all species of life [43], are medi-
ated by direct antimicrobial activities, modulation of host immune
responses, or both. Although initially termed antimicrobial pep-
tides (AMPs) due to their ability to kill microbes directly, the
immunomodulatory properties associated with many of these pep-
tides have resulted in their designation as host defense peptides
hydrophobicity to allow for interaction with or traversing of mem-
branes of target cells [21,18].
HDPs are elements of the host innate immune defense sys-
tem. The upregulation and/or release of HDPs is induced upon
infection with bacteria or viruses. The more than 1000 naturally
occurring HDPs are divided into 4 broad structural classes, which
represent amphipathic ␣-helixes (e.g. cathelicidins), ␤-sheets with
2–4 disulfide bridges (␣ and ␤ defensins and protegrins), extended
structures (indolicidin), and beta-loop peptides (brevinin) [19].
Intensive research has led to the development of synthetic pep-
tides with enhanced antimicrobial activities based on their natural
counterparts as well as IDRs (innate defense regulators), which
do not need to kill microbes, but rather stimulate the host
immune defenses to facilitate clearance of an infection in vivo
[12,13,20,36].
∗
Corresponding author at: Department of Pathology and Laboratory Medicine,
HDPs have received increased interest as antimicrobial thera-
peutics, because of their antimicrobial activity against a variety
of pathogenic bacteria, including those that are resistant to
Emory University, Whitehead Research Bldg. #144, 615 Michael St., Atlanta GA
30322, United States.
E-mail address: dkalman@emory.edu (D. Kalman).
0196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2010.08.008

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B. Bommarius et al. / Peptides 31 (2010) 1957–1965
conventional antibiotics [2]. HDPs display minimal inhibitory con-
centrations (MIC) as low as 0.25 ␮g/mL in vitro [28], and their
capacity to engender resistance is lower than that of conventional
antibiotics. This likely occurs either because HDPs nonspecifically
interact with bacterial membranes or because they act on multiple
targets [13,19,41]. Nevertheless, constitutive or induced resistance
to HDPs has been reported in several pathogenic bacteria, and can
be recapitulated in vitro (for an overview see [22,32,41]).
Several groups have taken advantage of advances in modeling
and QSAR (Quantitative Structure Activity Relationships) methods
[9,33], as well as an understanding of how endogenous peptides
affect innate immune defenses [6,11,42], to develop synthetic HDPs
and IDRs not found in Nature. QSAR is a mathematical relation
between the biological activity of a molecular system and its
geometric and chemical characteristics. Most structure–function
studies provide a working conceptual model of bioactive models.
In summary, QSAR studies attempt to find a consistent relationship
between biological activity and molecular properties, so that these
“rules” can be used to evaluate the activity of new compounds.
Synthetic peptides with antimicrobial activity and/or
immunomodulatory capabilities have proven remarkably effective
when used in animal models of infection against diverse pathogens
including MRSA and malaria among others [1,5,30].
Despite their promise as therapeutics, clinical trials with
endogenous or synthetic HDPs have been limited, with none yet
approved for use in humans [2], although other peptide drugs have
been approved such as the HIV fusion inhibitor Fuzeon. One impor-
tant contributing factor is that the costs of manufacturing are high,
making the price per dose quite expensive. Current production
methodologies center on solid phase peptide synthesis, but require
expensive precursor components. Several reports have introduced
methods for producing HDPs in bacteria or yeast [8,17,26,31,44],
but to date, scale-up has not proven cost-effective. Even when pro-
ductionispossible, purification requires multiple biochemical steps
that are expensive and significantly reduce yield.
Here, we describe a procedure to produce HDPs on a large-scale
in bacteria. We have chosen seven cationic peptides for expression:
LL-37 (# 1) is a 37-amino acid human cathelicidin peptide,
which has strong immunomodulatory activity and weaker direct
antimicrobial activity due to inhibition by physiological salt con-
centrations. By contrast, CRAMP (# 2) is the mouse homolog
of human LL-37 with 60% identity and somewhat more potent
direct antimicrobial activity. IDR-1 (# 3) is the prototype of the
IDR peptide class since it has no direct antimicrobial activity but
protects in animal infections by modulating the innate immune
response and is now in Phase I clinical trials (seq: KSRIVPAIPVSLL)
[36].
MX-226 (# 4) is the most clinically advanced antimicrobial pep-
tide that has shown statistically significant efficacy in Phase III
clinical trials as an antimicrobial and in Phase II trials as an anti-
inflammatory.
E5 (also called sub2, # 5) and E6 (also called sub3, # 6) are
12-amino acid peptides optimized through substitutions into the
bovine bactenecin peptide [15] while HHC-10 (# 7) is a promising
broad spectrum 9-amino acid candidate that emerged from a QSAR
modeling approach [9].
2. Materials and methods
2.1. Bacterial strains and plasmids
Escherichia coli strain BL21 (DEplys) was used as a host for
cloning and gene expression of various smtp3-HDP fusion con-
structs. E. coli was grown at 37 ◦C in LB media for cloning and at
30 ◦C in LB media for expression; kanamycin (25 ␮g/mL) was added
during growth of plasmid containing strains. The high-density fer-
mentation media was prepared as described [24].
The vector pET28b was used for cloning and expression of the
target genes. The vector allows for expression of exogenous pro-
teins and peptides under the control of the T7 promoter (Novagen,
Madison, WI). Sumo-pET28a and sumoase-pET28b were kindly
provided by Dr. X. Cheng (Emory University). All recombinant DNA
manipulation was performed using standard protocols.
2.2. Construction of expression vectors for expression of cationic
peptide fusions
The overall scheme for construction of SUMO-HDP fusion pro-
teins is illustrated in Fig. 1A. Briefly, the yeast smtp3 gene, encoding
sumo, including a 5ꢀ 6xHis-linker was cloned into pET28a using the
Nco1 and Nde1 sites. Peptide sequences are presented in Fig. 1C.
The smtp3 gene was modified and shortened at the 3ꢀ-end to allow
for a seamless fusion product after the Gly–Gly target sequence
(permitting release of the peptide by sumoase Ulp1) using primers
listed in Table 1.
All chosen HDP genes encode the active form of the correspond-
ing peptides. The resulting PCR product fuses the cationic peptide
gene 3ꢀ in frame with the GG cleavage site of smtp3 (sumo), leaving
no residual amino acids after cleavage (Fig. 1A). The PCR product
was restricted and ligated into the restricted vector, generating his-
sumo-hdp-pET28b. For LL-37 and CRAMP, the corresponding genes
were cloned from a cDNA isolated either from human bone mar-
row cDNA library (LL-37) or from primary mast cells harvested from
C57Bl/6 mice (data not shown). Starting from the cDNA, the active
part of the gene for LL-37 was isolated via 2-step PCR amplification
protocol with primers 1, 7, 8 and 9. The resulting PCR construct
was cloned into pET28b using the generated Nco1-Nde1 fragments,
and E. coli BL21 (DElys, RIL) were transformed with the ligated con-
struct. Positive clones were selected after colony PCR analysis, and
tested for expression.
2.3. Expression and high-density fermentation of positive strains
For small-scale expression, 5 mL overnight cultures of E. coli cells
(BL21 DE(plys)) harboring the vector sumo-AMP/HDP-pET28a were
incubated for 12 h at 37 ◦C. Subsequently 2 mL were used to inoc-
ulate 500 mL LB supplemented with kanamycin (25 ␮g/mL). The
cultures were incubated at 30 ◦C in shaking flasks, and expression
was induced at an OD600 of 0.5 with IPTG at a final concentra-
tion of 0.4 mM. Cells were harvested 4 h after induction and lysed
in PBS + 300 mM NaCl + 10 mM imidazole using sonication. Lysed
extract was cleared by centrifugation at 13,000 × g for 20 min.
Large-scale production of fusion protein was achieved using high-
density fermentation in a 10 L fermentor with high-glucose feed as
described previously [24,39].
The technique for HDP production in bacteria relies on gener-
ating a fusion protein between the peptide and SUMO that both
protect the bacteria from the toxicity of the peptides, and the pep-
tides from host proteolytic enzymes. The sumoase protease Ulp1,
which specifically cleaves after the C-terminal Gly–Gly residues in
SUMO is then used to release HDP sequences at the C-terminus
of the fusion protein [4,27,38]. Finally, we have devised a simple
two-step purification protocol. The procedure is readily amenable
to cost-effective industrial-scale GMP production of HDPs and
IDRs.
2.4. Purification of expressed fusion proteins
The catalytic domain of sumoase was purified as described pre-
viously [27] and frozen as concentrated 50% glycerol stocks in
the same concentration as described. The enzyme is very specific
for SUMO, and no nonspecific activity was observed; depending
on the volume of the sample, 5–10 ␮L of sumoase, corresponding

B. Bommarius et al. / Peptides 31 (2010) 1957–1965
1959
Fig. 1. (A) Schematic outline of the cloning strategy for in frame translation of the fusion product SUMO-peptide. Enhanced is the amino acid sequence at the cleavage site,
showing the GG recognition sequence for sumoase followed by a generic peptide sequence. (B) Schematic outline of the purification protocol after expression of the fusion
protein. Purification is achieved with 2 chromatographic steps; the cationic exchange chromatography is optional if a large RPC column size is less desired and cost-restrictive.
(C) Amino acid sequence and the corresponding mass (Da) for all the peptides tested with this methodology.
to 400 U, were added for complete cleavage of the fusion pro-
tein.
acetonitrile (Fig. 1B). Purification was monitored using tricine-SDS-
PAGE [35].
After cell lysis by sonication, the cleared supernatant was
applied to
a Ni-NTA gravity column and allowed to pass
2.5. Characterization of the purified recombinant peptides
through. The column was washed with 10× column volumes of
PBS + 300 mM NaCl + 20 mM imidazole and subsequently with 10×
column volumes of PBS + 300 mM NaCl + 40 mM imidazole. Bound
proteins were eluted with 2–3 column volumes of PBS + 300 mM
NaCl + 250 mM imidazole.
Purified peptides were lyophilized and compared to the syn-
thetic peptides using matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF) to assess the cor-
rect size after cleavage and Edman degradation to verify the
correct amino acid content. All MALDI-TOF spectra were col-
lected in positive ion mode and the collected data was within the
acceptable accuracy of the instrument’s capabilities. For the antimi-
crobial peptides, MICs were determined in vitro using the modified
microtiter broth dilution method [40] and compared to established
The eluted fusion protein was cleaved either overnight at 4 ◦C
or for 1 h at RT using 400 U of sumoase and the cleaved peptide fur-
ther purified and separated from its fusion partner using a C2/C18
or RESOURCETM reversed phase chromatography column and a
0.1%TFA/acetonitrile gradient; peptides eluted between 25 and 35%
Table 1
Primer sequences for all the HDP genes cloned and fused to smtp3 (sumo). The restriction sites are shown in bold, the HDP gene sequence
is shown in lower case and the smtp3 (sumo) fusion part is underlined.
Primer name
Primer sequence
5ꢀ-cgcgCCATGGGGcatcatcatcatcatcatTCGGACTCAGAAGTC-3ꢀ
5ꢀ-His-sumo-Nco1s
3ꢀ-Idr1-Ndeas
5ꢀ-GCGCGCCCATATGctacagcagggacaccgggatcgccggcacgatgcgggatttCACCAATCTGTTC-3ꢀ
5ꢀ-GCGCGCCCATATGctaccagcggatccatttccaccagcgtttACCACCAATC TGTTC-3ꢀ
5ꢀ-CGCGCCCATATGctactagcggcggacgcggatgacgacgatgcgccagcggcgACCACCAATCTGTTC-3ꢀ
5ꢀ-CGCGCCCATATGctactagcggcggacgcggatgacgacgatgcggcggaggcgACCACCAATCTGTTC-3ꢀ
5ꢀ-GCGCGCCCATATGctatttgcggcgccacggccaccacggccagcgaaggatACCACCAATCTGTTC-3ꢀ
5ꢀ-GAACAGATTGGTGGTctgctgggtgatttcttccgg-3ꢀ
3ꢀ-HHC10-Ndeas
3ꢀ-sub3Ndeas
3ꢀ-sub2Ndeas
3ꢀ-Mx226Ndeas
5ꢀ-sumoLL37linker
3ꢀ-sumoLL37linker
3ꢀ-LL37Ndeas
5ꢀ-ccggaagaaatcacccagcagACCACCAATCTGTTC-3ꢀ
5ꢀ-GCGCGCCCATATGctaggactctgtcctgggtac-3ꢀ

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B. Bommarius et al. / Peptides 31 (2010) 1957–1965
Fig. 2. (A) Fermentation profile of a 10 L high-density fermentation of SUMO-IDR-1 using glucose feed and IPTG induction for 14 h. The fermentation yielded 1.5 kg biomass.
(B) Comparison of the RP-chromatography profile for both the synthetic (upper panel) and recombinant IDR-1 peptide (lower panel). Both peptides eluted at the same percent
of acetonitrile. (C) MALDI analysis of the purified IDR-1 peptide as well as the remaining his-SUMO protein after cleavage. The determined mass for IDR- 1 was identical to
the synthetic peptide and the correct mass for his-sumo revealed 100% cleavage using its protease sumoase. The MS profile for IDR-1 was magnified for better visualization.
(D) Tricine-SDS-PAGE separation of the proteins from the recovered fractions from RP-chromatography shown in B. As can be seen in lanes 1 and 2, pure IDR-1 was recovered
from fractions 11 and 12 followed by elution of SUMO in lanes 3–10. Lane 5 shows the Kaleidoscope Marker Plus (BioRAD).
MICs for the corresponding chemically synthesized peptides. For
immunomodulatory peptides, induction of the cytokines TNF␣ and
IL6 as well as the chemokine MCP-1 were assessed by enzyme
linked immunoabsorbant assay (ELISA) following stimulation of
human peripheral blood mononuclear cells (PBMC) with peptides
for 16 h. PBMCs from different human donors were isolated as
previously described [29] in accordance with University of British
Columbia ethical approval and guidelines, and the cytokine induc-
tion was compared to the results with the synthetic peptide, as
described previously [36]. Peptide concentration at 300 ␮g/mL
was used in all of the ELISA assays. Bacterial contaminants were
removed using the ProteoSpin kit (Norgen) according to the man-
ufacturer’s guidelines.
Therefore, we concluded that the His-SUMO served to block the
antimicrobial activity of the fused peptides.
Several cationic peptides of different peptide lengths were cho-
sen for expression using the SUMO fusion system (Fig. 1C). These
included LL-37, CRAMP, MX226, IDR-1, E5 and E6 and HHC-10.
All constructs tested expressed high levels of the fusion pep-
tide both in small scale (50 mL) as well as 1 L expression levels
(data not shown). The identification of the intact fusion peptides
provided evidence that the SUMO-peptide fusion was protected
against cleavage by endogenous proteases. On small scale, increase
in peptide length did not influence the overall expression yield of
the fusion protein, but increasing peptide length will enhance the
yield of the pure peptide as a result of a more favorable ratio of
peptide to SUMO within the fusion construct.
3. Results
3.2. High-density fermentation
3.1. Expression of cationic peptides in bacteria
Two of the peptides, one immunomodulatory peptide (IDR-1)
and one antibacterial (E6) were chosen for high-density fermen-
tation with glucose feed in a 10 L pilot scale. We decided on IDR-1
because it represents the prototype of the immunomodulatory pep-
tides [36] and has entered phase 1 clinical trial with Inimexpharma;
and on E6 as one of the first published short peptides resulting
from a bioinformatics approach [15]. Both fermentations yielded
around 1.5 kg wet biomass, of which 6% encompassed SUMO-IDR-
1 and 4.3% SUMO-E6 (Figs. 3B and 4D). Under the conditions used,
expression of IDR-1 appeared at near optimal levels based on the
We sought to develop a cost-effective means to express cationic
peptides in E. coli with high yield and then purify them. To express
peptides in E. coli, the cDNAs encoding them were cloned in frame
and 3ꢀ to a gene encoding His-tagged sumo. Whereas expression
of the genes alone under the same promoter control resulted in
few if any colonies (data not shown), adding the His-sumo to the
5ꢀ-end of the peptide gene resulted in normal bacterial growth
with concomitant fusion protein expression in the soluble fraction.

B. Bommarius et al. / Peptides 31 (2010) 1957–1965
1961
Fig. 3. (A) Cytokine release of PBMCs from different human donors after treatment with either recombinant or synthetic IDR-1. Cytokines tested are IL-6, TNF␣ as well as
the chemokine MCP-1. (B) Purification table for IDR-1 showing the yield of pure IDR-1 after an initial pilot purification of 10 g of biomass from the fermentation as well as a
projection of possible yields from processing the total biomass of this fermentation. (C) Tricine-SDS-PAGE comparing cleaved peptide yields of a high-density fermentation
against a 1 L shaking flask expression. The amounts shown for E5 and E6 are representative for all the peptides tested in shaking flasks expression.
fusion protein amounts present in the soluble fraction, whereas E6
did not. Although no bacterial lysis occurred during the fermen-
tation of either peptide, we anticipate that each construct would
likely require optimization to achieve maximum yield.
ples). As shown in Figs. 2 and 4, we achieved excellent purification
from the scale-up investigation of IDR-1 and E6. To do this, 10 g of
the harvested biomass from the fermenter was lysed, cleared and
applied to an affinity chromatography column in purification step
1 using Ni-NTA (Fig. 1B). The fusion protein was eluted from the
column with 250 mM imidazole and then cleaved with ∼400 U of
sumoase for 1 h at RT. SUMO-IDR-1 accounted for 6% of the total
protein produced, which gave an estimated yield of 0.48 g/L fer-
mentation (Figs. 2D and 3D). For peptides with high tryptophan
content, it was necessary to add 1 M urea to the cleavage reaction
to ensure precise cleavage of the peptide from the fusion partner
SUMO (Figs. 4B and 5A).
Following cleavage, proteins were successfully separated using
reversed phase chromatography to produce homogenously pure
peptide (Figs. 2D and 4C). RP-chromatography was needed to sep-
arate SUMO and sumoase from the peptide, because repeated
application onto an affinity Ni-NTA did not result in successful sepa-
3.3. Purification of peptides
In the first step, the His-SUMO fusion peptides were puri-
fied using a Ni-NTA sepharose column. The peptides were then
released from the fusion by cleavage with the SUMO-specific
protease sumoase, which recognizes the three-dimensional fold
around a GG sequence at the SUMO-peptide boundary (Fig. 1A). The
released peptide was further purified using RPC-C2/C18-FPLC or
RESOURCETM RPC-FPLC. A C8 linker on the RPC column was shown
not to be effective in separating sumo from the peptide.
Purification and cleavage of the peptide was successful for all
peptides tested on a small scale (see below for selected exam-

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B. Bommarius et al. / Peptides 31 (2010) 1957–1965
Fig. 4. (A) Fermentation profile of a 10 L high-density fermentation of SUMO-E6 using glucose feed and IPTG induction for 14 h. The fermentation yielded 1.3 kg biomass.
The total biomass is comparable to the one achieved using an immunomodulatory peptide, indicating that expression of an antimicrobial peptide is not deleterious to the
bacterial host. (B) Tricine-SDS-PAGE of the purification steps used to achieve pure E6. Lane 2 showing RPC fraction # 6 corresponds to the fraction sent off for MALDI (see
C). (C) RP-chromatography profile of the purification of E6 and the subsequent determination of its mass. MALDI analysis determined the correct mass of 1665 Da for this
peptide with no further contamination present up to 30 kDa. The MS profile of E6 was magnified for better visualization. (D) Purification table showing the overall yield for
E6 purification. (E) Direct comparison of MIC for both synthetic and recombinant E6 using E. coli K12 and P. aeruginosa PA014. The recombinant E6 showed identical MIC for
E. coli and a slightly higher MIC for P. aeruginosa, again indicating that no contaminant protein is present, since then the amount of E6 would be lower than the one for the
synthetic peptide.
ration of the two (data not shown). Our data suggest that the overall
positive charge of the peptide (between +4 and +6) may induce
electrostatic interactions with negatively charged residues within
SUMO (overall charge −5), perhaps annealing the two proteins dur-
ing a non-hydrophobic separation procedure. Indeed it is likely that
these interactions play an important role in both neutralization of
biological activity and protection from endogenous proteases.
as a fusion and successful cleavage with sumoase and purification
achieved. 1 M urea was required to ensure correct cleavage at the
desired position on SUMO (Fig. 5A), a procedure found necessary
for Trp-containing peptides. We also successfully expressed and
purified larger HDPs including CRAMP and LL-37 (36 and 37 aa long)
with this methodology (data not shown).
3.5. Identification of purified peptides
3.4. Expression and purification of other peptides
The identity of the isolated peptides was confirmed by mass
spectrometry (Figs. 2C and 4C) using MALDI-TOF to determine the
exact mass of purified peptides. IDR-1 showed the exact molecular
Two other cationic peptides, HHC-10 (Fig. 5A) and MX-226 (data
not shown), were successfully expressed in shaking flask cultures

B. Bommarius et al. / Peptides 31 (2010) 1957–1965
1963
available equipment, a total of 5 runs were needed to process the
10 g biomass obtained from a single purification using the C2/C18
RPC column. However this was reduced to 2 runs using the Resource
RPC column and therefore can readily be adapted to larger scale. To
determine yield, the purified peptide was weighed on a fine scale
after lyophilization. However, these measurements can only be
considered a rough estimate, since residual salt concentrations left
attachedtothe peptidecould affectthe weight. IDR-1concentration
was subsequently determined using amino acid analysis of the pure
peptide and showed about 50% residual salt within the peptide,
which is similar to synthetically produced peptides. Overall, 3 mg
of pure IDR-1 was obtained from 10 g of biomass (Figs. 2D and 3B).
In an optimized process, we expect to achieve 0.08 g/L fermenta-
tion of pure IDR-1. For E6 the purification of the fermented biomass
yielded 0.6 mg from 10 g wet cells (Fig. 4D). Despite similar fermen-
tations the heterologous expression of sumo-E6 only accounted for
4.3% of the total protein in E. coli. Tricine-SDS-PAGE analysis of E6
indicated several bands around 10 kDa, but thorough mass analy-
sis up to 30 kDa did not reveal any peak other than the one for E6.
These data suggest that significant aggregation might be occurring.
Alternatively, LPS may have become attached to the E6 peptide,
since the MW for LPS in E. coli is around 5–10 kDa.
3.7. Characterization of the IDR-1, E6 and HHC-10
For the HDPs E6 and HHC-10 we determined the mini-
mal inhibitory concentration (MIC) for growth of E. coli and
Pseudomonas aeruginosa PA014, and compared these values
to those obtained with the corresponding synthetic peptide
(Figs. 4E and 5C). Using both bacteria, MICs for the recombinant,
homogenous peptide E6 were identical to the corresponding syn-
thetic peptides, given that MICs are operatively considered to be
accurate to within twofold.
Fig. 5. Tricine-SDS-PAGE of the purification steps used to achieve pure HHC-10 from
a 1 L shaking flask expression, fraction 12 in lane 2 corresponds to the sample used
for mass determination. Again, no larger protein contaminants could be found, indi-
cating that the higher bands visible in the gel are possibly aggregates of HHC-10,
which could be avoided in a different solvent. (B) MALDI profile for HHC-10 show-
ing the correct mass of 1444 Da. (C) Direct comparison of MIC for both synthetic and
recombinant HHC-10 using E. coli K12 and P. aeruginosa PA014. The recombinant
HHC10 showed a slightly higher MIC for E. coli and P. aeruginosa.
For the immunomodulatory peptide IDR-1 the efficacy of the
recombinant peptide was compared to the synthetic peptides using
cytokine/chemokine ELISAs to determine the stimulation of a pro-
inflammatory response on PBMC from a variety of human donors.
TNF␣ was used as an indicator of inflammatory cytokine stim-
ulation and possible contamination with bacterial components,
because synthetic IDR-1 does not trigger a TNF␣ response. Initially,
the recombinant peptide yielded higher values for IL-6 and TNF␣
release than two different lots of synthetic peptide. This difference
could not be attributed to differences in apparent concentration
between the purified and synthetic peptides. We concluded that a
bacterial contaminant was still present in the IDR-1 preparation,
although the amount varied strongly with the donor. We there-
fore further purified the recombinant peptide using a LPS removal
kit (Norgen), successfully removing LPS as the contaminant and
achieving the same TNF␣ response for the recombinant peptide
compared to the synthetic peptide (Fig. 3A). On the other hand,
the IL-6 response and production of MCP-1 as an indicator for
chemokine induction was still higher between the recombinant and
synthetic IDR-1 (Fig. 3A), so a synthetic control IDR-1 peptide lack-
ing amidation (IDR-1-COOH) was included and indicated that the
increased MCP-1 and IL6 responses were due to the lack of amida-
tion on the recombinant peptide. Most synthetic peptides include a
C-terminal amidation as part of the synthesis process, but bacteria
cannot produce peptides with a C-terminal amidation, thus post-
translational modifications of bacterially produced peptides via a
chemical route might be necessary for therapeutic development.
weight (MW) of 1391.9 Da, which corresponded to its theoretical
mass (cf. Figs. 1C and 2C). N-terminal sequencing of the IDR-1 in
the gel confirmed the correct first 5 amino acids of IDR-1 (KSRIV,
data not shown), indicating that cleavage occurred at the right
position after the Gly–Gly sequence in SUMO with no additional
amino acids introduced to the peptide. The other band observed
(Fig. 2C) at a molecular mass of 11,918.7 Da, was identical to the
mass of the separated SUMO recovered from reverse phase chro-
matography (see Fig. 2C, second peak) suggesting that our sumoase
cleavage protocol achieved 100% cleavage [34]. Likewise, the mea-
sured mass of 1664.83 Da for E6 corresponded to its theoretical
peptide mass (Fig. 4C, see Fig. 1C for comparison). Mass deter-
mination for the short, Trp-rich peptide HHC-10 after purification
showed the predicted mass of 1443.69 Da (Figs. 1C and 5B). In this
case, as mentioned above, correct mass of the purified, cleaved pep-
tide could only be achieved through addition of 1 M urea to help
unfold the short hydrophobic stretch of this Trp-rich peptide adja-
cent to the cleavage site for sumoase. Although sumoase is thought
to be highly specific [27,34], we have observed that introducing
a stretch of several tryptophan residues close to the cleavage site
most likely prevents access to the site through steric hindrance and
causes random cleavage within the fusion protein. Addition of 1 M
urea prevented such unspecific cleavage and led to the release of
the full-length peptide.
3.6. Peptide yields
4. Discussion
The amount of IDR-1 shown in Fig. 2D corresponds to 1% of
the total IDR-1 recovered from a single chromatography run. With
To explore the pharmaceutical and therapeutic potential of
antimicrobial peptides, a cost-effective and scalable method for

1964
B. Bommarius et al. / Peptides 31 (2010) 1957–1965
production of active and effective HDPs is required. Procedures
to express HDPs as recombinant peptides have encountered dif-
ficulties associated with cytotoxicity to the bacterial host, and,
as a consequence, difficulty in scale-up and low yields following
purification [8,31,44], especially when large fusion proteins are
chosen [16]. Even when successful, the requirement for processing
of fusion peptides (often requiring chemicals or costly enzymes)
and multistep purification of peptides has shown reduced yields,
has not proven cost-effective, and has met with intermittent suc-
cess depending on the peptide.
In this study we have described a procedure for high yield pro-
duction of several antimicrobial or immunomodulatory peptides
using a fusion protein partner that has been well established to
ensure high-level soluble expression of fusion proteins, even with
proteins that are otherwise difficult to express (e.g. MMP13 [27]).
Using this system, we have found that the fusion protein accounts
for 10–25% of total protein, and yielded 3 mg of pure peptide from
10 g of biomass.
Unlike previous methods, expression of the SUMO-peptide
fusion does not have to be forced into the insoluble fractions,
which, on an industrial scale, can be costly due to the requirement
for urea and guanidium chloride to solubilize the inclusion bod-
ies [14,23,46]. Moreover, the fusion protein remains in the soluble
fraction without lethal effects to the host bacterial cells. Thus, pro-
teolytic degradation of the fusion protein and release of the peptide
does not readily occur. Generally, there are several options available
to cleave a peptide from its fusion partner, utilizing both chemical
and enzymatic routes of cleavage. For chemical cleavage, two differ-
ent methods have been explored. Cyanogen bromide, which cleaves
C-terminally after methionine, has been used extensively, but is
inefficient in its cleavage [14]. Second, cleavage of the acid-labile
peptide bond between asparagines and proline using hydrochloric
acid [46], leaves a proline overhang at the N-terminus of a peptide
and requires neutralization of the acid, properties that are neither
generally nor easily applicable on an industrial scale.
and MX-226 (www.migenix.com)), whereas other recent publica-
tions have achieved expression and purification of peptides in the
range of 30 amino acids and more [7,25].
Our data also indicate that posttranslational modifications of
peptides can alter their cytokine profile, as demonstrated in Fig. 3A.
Amidation of the C-terminus of IDR-1, as it is common practice
for synthetic peptides, resulted in less cytokine response than the
free C-terminus IDR-1 samples, both synthetic and recombinant.
Peptide amidation can occur in mammals, since most hormonal
peptides and neuropeptides are amidated through peptidylglycine
␣-amidating monooxygenase PAM, which is essential for their
activity [3,10]. Amidation also protects from C-terminal peptide
degradation, which prolongs the half-life of peptides in serum.
So far, amidation has not been reported for endogenous human
antimicrobial peptides.
Correct determination of peptide concentration following
chromatography can only be achieved through amino acid determi-
nation, because salts used in the purification tend to attach to the
peptides. This is especially crucial when using cationic exchange
chromatography, where high concentrations of sodium chloride are
used to elute the peptide. We have found salt to be present in the
lyophilized peptide even after RP-chromatography as the last step,
which does not involve salt.
Taken together, this expression system allows for large-scale
production of HDPs, which retain activity similar to peptides
synthesized by chemical means. The system can produce both
immunomodulatory and antimicrobial peptides, apparently inde-
pendent of amino acid sequence, length, or charge in industrial
scale quantities. Even small peptides of just 9 amino acid in length
can be produced with acceptable yields. Purification is achieved
in two steps, which are easily scalable for industrial application.
In short, we provide evidence that this system will enable cost-
effective production of HDPs under GMP conditions in support of
therapeutic applications for these molecules against infectious dis-
eases.
Enzymatically, several proteases, such as thrombin, Factor Xa or
enterokinase, will cleave at their recognition sites, once these are
introduced within the linker between the fusion protein and the
peptide during cloning [45]. However, these enzymes are expensive
and not feasible for industrial scale purification. Sumoase is unique
in that it recognizes only residues within SUMO as its cleavage sub-
strate and cleavage occurs precisely after the Gly–Gly in the SUMO
sequence [27], leaving no unwanted amino acids at the N-terminus
of the peptide. Another important advantage is that this protease is
produced cheaply using the T7 driven pET system and can be easily
purified with Ni-NTA affinity chromatography in a single step [27].
Also, 400 U of enzyme were used for complete cleavage [27], which
seems substantial, but a 0.5 L culture yields 10,000× that amount,
suggesting that the enzyme is actually quite cost-effective.
A few labs have recently used the SUMO fusion system for cost-
effective antimicrobial peptide expression and demonstrated its
efficacy [7,25], however, we have shown that successful endotoxin
removal is critical when considering their use for therapeutic pur-
poses. Removal of endotoxins has proven to be a challenge and
several routes had to be taken until the unfavorable TNF␣ response
was prevented. Our data indicates that the bacterially produced
peptides have to be an exact match to the established synthetic
peptides, if they are to be successful therapeutics. As we have
demonstrated, this is especially true for the peptides’ immunomod-
ulatory potential, a peptide property that is gaining more and more
importance in therapeutic applications [2]. We were able to pro-
duce peptides of varying lengths ranging from 37 amino acids for
LL-37 down to 9 amino acids for HHC-10 with excellent MIC values.
To the best of our knowledge, nobody has achieved bulk production
of such small peptides in bacteria, especially not peptides that are
already tested in clinical trials (IDR-1 (www.inimexpharma.com)
Acknowledgements
We appreciate the financial support from the Foundation for
the National Institutes of Health and Canadian Institutes for Health
Research through the Grand Challenges in Global Health Initia-
tive to REWH. REWH is a recipient of a Canada Research Chair
while JK hold a fellowship from the Canadian Cystic Fibrosis
Foundation.
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