EXPRESSION AND CHARACTERIZATION OF ANTIMICROBIAL PEPTIDE
GAMBICIN FROM Culex quinquefasciatus IN Pichia pastoris
Phanthila Sirichaiyakul1,*, Rungarun Suthangkornkul1, Apanchanid Thepouyporn1,
Okabayashi Tamaki2, Yoshiharu Matsuura3, Naokazu Takeda4, Kazushi Motomura4
Dumrongkiet Arthan1,#
1
Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol
University, Bangkok, 10400 Thailand. *Master of Science in Tropical Medicine
(International Program), Thailand
2
Mahidol-Osaka Center for Infectious Diseases (MOCID), Faculty of Tropical Medicine,
Mahidol University, Bangkok, 10400 Thailand
3
Department of Molecular Virology, Research institute for Microbial diseases, Osaka
University, Osaka, 5645-0871 Japan
4
Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections
(RCC-ERI), Nonthaburi, 11000 Thailand
*e-mail: fairy_alizia@hotmail.com, #e-mail: dumrongkiet.art@mahidol.ac.th
Abstract
Antimicrobial peptides (AMPs), which can be found in many organisms, are generally
low molecular weight peptides containing less than 100 amino acid residues. Due to
ineffective conventional antibiotic treatments causing numerous mortalities of infected
patients in the world, AMPs are an interesting approach to the development of novel
therapeutics against pathogens, particularly antibiotic-resistant bacteria, because they are
safe, non-toxic to mammals, no bacterial resistance and showing broad-spectrum
antimicrobial activity. While the mosquito AMPs, namely cecropin and defensin have been
studied in depth, gambicin has not. Interestingly, an antimicrobial peptide gambicin isolated
from Anopheles gambiae cell lines possess antimicrobial activity against gram-positive and negative bacteria, filamentous fungi, and Plasmodium species. The isolation gambicin of
Culex genus and expression of recombinant mosquito gambicins remain unreported. This
study aimed to express recombinant Culex quinquefasciatus gambicin in Pichia pastoris
expression system. We successfully cloned and expressed the Culex quinquefasciatus
gambicin in Pichia pastoris. SDS-PAGE revealed obvious two protein bands with a
molecular weight of about 7.0 kDa, indicating high production of recombinant Culex
quinquefasciatus gambicin. Testing of antimicrobial activity against Escherichia coli by agarwell diffusion assay exhibited no activity. For further studies, a refolding approach to the
recovery of proper folding of 3-D structure of the active Culex quinquefasciatus gambicin is
required. Additionally, the expression of recombinant Culex quinquefasciatus gambicin in the
baculovirus expression system is an alternative method to obtain the active recombinant
Culex quinquefasciatus gambicin peptide.
Keywords: gambicin, antimicrobial peptides (AMPs), mosquitoes, Pichia pastoris, agar-well
diffusion assay
Introduction
The emergence of antibiotic-resistant pathogens has become a significant global
problem, resulting in ineffective conventional antibiotic treatments [1]. Antimicrobial
peptides (AMPs) are low molecular weight peptides found in many organisms, which
generally consist of less than 100 amino acid residues [2,3]. AMPs exhibit broad-spectrum
antimicrobial activity, are safe, non-toxic to mammals, and indicate no bacterial resistance
[4,5]. The mechanism underlying AMPs’ action against pathogens is based on the alteration
of the biophysical properties of membranes, leading to ion and metabolite leakage from cells,
the result of AMPs’ interference with the phospholipids integrity of the cell membrane [6].
Thus, AMPs are of great interest for the development of alternative therapeutic drugs [7].
Presently, AMPs and their derivatives have reportedly been used in many applications
including plants and agricultures, food industries, aquaculture, animal husbandry, and drug
developments [6,8].
While the mosquito AMPs, cecropin and defensin, have been studied in depth [9,10,11],
gambicin has not. Gambicin, isolated from Anopheles gambiae, has been found to possess
antimicrobial activity against some gram-positive and -negative bacteria, filamentous fungi,
and Plasmodium species [12]. However, the isolation gambicin of Culex genus, recombinant
mosquito gambicin production, to test broad-spectrum antimicrobial activity, remains
unreported. Since the preferred habitat for mosquitoes Culex genus is polluted water with an
abundance of bacteria and microbes [13], it was hypothesized that Culex gambicin may have
more effective antimicrobial activity than those from other mosquito gambicins. Thus, it is of
great interest in production of Cx. quinquefasciatus gambicin (CQ gambicin) to test broadspectrum antimicrobial activity.
This study is aimed to produce recombinant CQ gambicin in Pichia pastoris,
characterize its biochemical properties, and test it for antimicrobial activity.
Methodology
1. Mosquito samples
The fed female Cx. quinquefasciatus were provided by the Department of Medical
Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
2. Primer designs
The specific-gene primers were designed from the sequence of Cx. pipiens
quinquefasciatus salivary gambicin gene (Accession No. AY388563.1). Forward primer
without signal-peptide sequence and reverse primer were included the restriction enzyme
XhoI and an XbaI sites, respectively (Table 1).
Table 1. The specific-gene primers and applications.
Primer names
CQ_Gam-XhoI-pZb_F
CQ_Gam-XbaI-pZb_R
Sequences (5’-3’)
GGG GGG CTC GAG AAA AGA GAG GCT
GAA GCT TGG GTG TAT GTC TAT GCG
AAA ACC TGC
GGG GGG TCT AGA TCA CCC AAT GAA
GCA CTC GGT AAT GTA ACG
Application
Amplification of
gambicin gene
Amplification of
gambicin gene
3. Total RNA extraction
Female Cx. quinquefasciatus mosquitoes were knocked by freezing at -20oC followed
by grounded with liquid nitrogen to obtain the fine powder. To extract total RNA, TRIZOL
reagent (Invitrogen, USA) was added. All steps of total RNA extraction were performed
according to the instruction manual of Invitrogen (USA). Total RNA extracts were
subsequently used as a template for the first-strand cDNA synthesis.
4. Reverse transcription-Polymerase Chain Reaction (RT-PCR)
The first-strand cDNA was synthesized according to manufacture protocol
(Invitrogen, USA). To synthesize the first-strand cDNA, total RNA extracts about 1 µg was
reverse-transcribed by SuperScriptTM III reverse transcriptase cDNA and oligo dT12-18 as a
primer. To amplify CQ gambicin gene, the first-strand cDNA was used as a template for PCR
reaction containing 1X PCR buffer, dNTPs, Mg2+, specific-gene primers and KOD Hot Start
DNA Polymerase (Novagen®). The PCR condition was set as follows: 1 cycle of 95oC for 2
min; 30 cycles of 95oC for 20 sec, 62oC for 30 sec, 70oC for 30 sec; and 1 cycles of 70oC for
3 min. The PCR product was analyzed by 1.7% agarose gel electrophoresis, excised, and gelpurified by Gel/PCR DNA Fragments Extraction Kit (Geneaid, USA).
5. Construction of recombinant expression plasmid harboring gambicin gene
The fragment of gambicin gene was cloned into pPICZαB vector at downstream of
the α-factor signal peptide sequence. Both of purified CQ gambicin PCR product and
pPICZB vector (Invitrogen) were digested with XhoI and an XbaI (TAKARA), and
followed by ethanol precipitation, and ligation by T4 DNA ligase (Promega) according to the
supplier’s instruction manual (Promaga). Transformation of ligation products into
Escherichia coli DH5α, selection of positive clones by LB agar plate containing Zeocin and
colony PCR techniques, plasmid extraction and DNA sequencing were carried out to obtain
the recombinant pPICZαB plasmid harboring gambicin gene.
6. Transformation the expression plasmid into P. pastoris competent
Transformation the expression plasmid into P. pastoris competent was performed
according to the instruction manual of Invitrogen. Briefly, not only the recombinant
pPICZB plasmid harboring CQ gambicin but also the pPICZB vector were digested with
SacI (TAKARA) to make a linear form of DNA, and then transformed into P. pastoris strain
GS115 competent cells. Positive transformants were screened from YPDS (1% yeast extract,
2% peptone, 2% dextrose, 1M sorbitol, 2% agar) plates containing Zeocin at a final
concentration of 150 μg/ml.
7. Methanol-induced protein expression in recombinant P. pastoris
Expression of the recombinant protein in P. pastoris was performed according to the
manufacture protocol (Invitrogen). To produce CQ gambicin peptide, both transformant
harboring CQ gambicin and pPICZB empty vector were cultured in BMGY (1% yeast
extract; 2% peptone; 100 mM potassium phosphate, pH 6.0; 1.34% YNB; 4 x 10–5 % biotin;
1% glycerol) until A600 reading at 5. The cell pellets were harvested and resuspended in
BMMY (1% yeast extract; 2% peptone; 100 mM potassium phosphate, pH 6.0; 1.34% YNB;
4 x 10–5 % biotin; 0.5% methanol). Subsequently, the cell solution was further cultured in a
shaking incubator at 250 rpm and 16C. Since P. pastoris is methylotrophic yeast, methanol
used for induction of expression was added to each cell suspension in BMMY at a final
concentration of 0.5% every 24 hrs to maintain induction. After 5 days, the supernatants were
harvested by centrifugation and kept at 4 C until used.
8. SDS-PAGE analysis
SDS-PAGE was performed with 4% stacking gel and 15% resolving gel according to
Laemmli et al [14].
9. Internal protein sequence analysis
The internal protein sequence analysis was performed by in-gel digestion with trypsin
and identified by LC-MS/MS at Salaya Central Instrument Facility (SCIF), Mahidol
University. The mass spectrometric data were analyzed by mascot MS/MS ions search engine
(http://www.matrixscience.com/).
10. Antimicrobial activity assay
The antimicrobial activity was determined by agar-well diffusion method. E. coli, a
representative of bacteria, was used for antimicrobial test. Briefly, E. coli was cultivated on
nutrient agar at 37C for 18 hours, subsequently inoculated in the nutrient broth. It was
cultured at 37 C, 250 rpm for 3-5 hours. The cell numbers were adjusted to 0.5 McFarland
standards by using 0.9% normal saline solution. These cultured medium was streaked onto
Mueller-Hinton agar plate for 3 dimensions and let it dry for 5 minutes. The wells were done
by punching with a diameter of 5 mm. All tested solutions and ampicillin as a positive control
were filled in agar wells. The agar plate was incubated at 37C for 18-24 hours. The
minimum inhibitory concentration (MIC) is defined as the lowest concentration of the
recombinant CQ gambicin in agar plates illustrating visible inhibition zone.
Results
1. Construction of recombinant expression plasmid
The size of the PCR product was about 250 bps, which correspond to mosquito
gambicin gene, indicating that CQ gambicin gene was successfully amplified, as shown in
Figure 1. The purified PCR product was then constructed into expression vector pPICZB.
DNA sequences of two selected positive clones are identical (data not shown). Based on
protein alignment, the deduced amino acid sequences of recombinant CQ gambicin shows
100%, 85%, and 73% similarity to those of Cx. pipiens quinquefasciatus (AAR18451.1), An.
gambiae (ACA05602.1), and Ae. aegypti (AAL76025.1) gambicin genes, as seen in Figure 2.
Figure 1. The PCR product of CQ gambicin gene amplification.
Lane 1: DNA marker; Lane 2: CQ gambicin gene; Lane 3: Negative control of the reaction.
Figure 2. The alignment of the deduced amino acid sequences of gambicin peptides without signal peptide
among CQ gambicin (CQ), Cx. quinquefasciatus (Cx.pq; AAR18451.1), Ae. aegypti (Ae.a; AAAL76025.1) and
An. gambiae (An.g; ACA05602.1).
2. Expression and Characterization of recombinant CQ gambicin
A clone of P. pastoris expressing the highest recombinant CQ gambicin as judged by
SDS-PAGE was cultured in the optimal BMMY, 0.5% methanol expression conditions and
compared with a clone of P. pastoris harboring pPICZB as a control. SDS-PGAE analysis
of the supernatant of a control (Figure 2; lane 1) and a clone of P. pastoris harboring a CQ
gambicin (Figure 2; lane 2) revealed no band and two bands of protein at molecular weight
about 7 kDa, respectively. The tryptic digestion of two protein bands on SDS-PAGE was
identified by LC-MS/MS. The peptide sequences, observed mass per charge ratios, and
molecular weight of the peptides of upper and lower bands were shown in Table 2 and 3,
respectively. Ions scores of identical peptides were above 95% confidence. The mass per
charge ratios of peptides were subsequently analyzed using Mascot software. These results
showed that 5 and 4 tryptic peptide sequences of upper and lower bands were partially
identical to deduced amino acids of CQ gambicin. The percents of coverage of matched
protein sequence to the upper and lower bands identified by this method were 63 and 52,
respectively.
Figure 3. SDS-PAGE analysis of supernatants obtained from clones of P. pastoris harboring
empty pPICZaB vector (lane1) and CQ gambicin gene (lane2).
Table 2. Peptide mass fingerprint of the tryptic-digested recombinant CQ gambicin (upper band)
identified by LC-MS/MS.
Observed
m/z
Mr (expt)
Mr (calc)
Ions score
Expect
442.1888
882.3631
882.3654
56
0.41
501.7330
1001.4541
1001.4528
26
5.4e+02
521.7341
1041.4537
1041.4549
62
0.11
606.7866
1211.5586
1211.5605
68
0.027
447.5581
1339.6525
1339.6554
44
6.8
Peptide
R.FGTCQDR.Y +
Carbamidomethyl (C)
R.YITECFIG.- +
Carbamidomethyl (C)
R.NCGYGSLGSK.K +
Carbamidomethyl (C)
K.YVSCDGATAIR.N
+ Carbamidomethyl (C)
K.KYVSCDGATAIR.N
+ Carbamidomethyl (C)
Table 3. Peptide mass fingerprint of the tryptic-digested recombinant CQ gambicin (lower band)
identified by LC-MS/MS.
Observed
m/z
Mr (expt)
Mr (calc)
Ions score
Expect
442.1889
882.3633
882.3654
53
0.74
522.2256
1042.4366
1041.4549
64
0.083
606.7868
1211.5590
1211.5605
58
0.3
447.5580
1339.6523
1339.6554
22
1e+03
Peptide
R.FGTCQDR.Y +
Carbamidomethyl (C)
R.NCGYGSLGSK.K +
Carbamidomethyl (C)
K.YVSCDGATAIR.N +
Carbamidomethyl (C)
K.KYVSCDGATAIR.N
+ Carbamidomethyl (C)
3. Antimicrobial activity assay
The recombinant CQ gambicin peptide was tested for antimicrobial assay against E.
coli by agar-well diffusion. As a result, there was no any inhibition zone of the recombinant
CQ gambicin in plate comparing to ampicillin as s positive control (Figure 4).
Figure 4. The result of agar-well diffusion assay intimating that no inhibition zone (indicating at arrow).
(A) recombinant CQ gambicin peptide, (B) pPICZαB (negative control),
(C) Ampicillin (positive control), (D) Sterile water (negative control).
Discussion and conclusion
The cDNA of CQ gambicin was cloned into pPICZαB by fusion gambicin without
signal peptide with the Saccharomyces cerevisiae alpha-mating factor propeptide sequence of
pPICZαB, and successfully expressed gambicin in P. pastoris culture medium, which is the
optimal BMMY, 0.5% methanol expression conditions. SDS-PGAE analysis of the
supernatant containing CQ gambicin revealed two bands of protein at molecular weight 7
kDa, corresponding to the size of the mature An. gambiae gambicin as reported previously
[12]. This result indicated high production of recombinant proteins. The tryptic digestion of
two protein bands on SDS-PAGE was identified by LC-MS/MS. Five and four tryptic peptide
sequences of upper and lower bands were identical to the corresponding peptides from
deduced amino acids of CQ gambicin. The percents of coverage of matched protein sequence
to the upper and lower bands identified by this method were 63% and 52%, respectively.
These results obviously indicate that two bands on SDS-PAGE are the recombinant CQ
gambicin successfully expressed in P. pastoris. However, two bands of recombinant CQ
gambicin found on SDS-PAGE are different from the purified An. gambiae gambicin, which
was shown a single band on SDS-PAGE [12]. This result suggested that the two recombinant
CQ gambicin peptides may cause by the proteolytic digestion. Additionally, the CQ gambicin
exhibited no anti-microbial activity against E. coli, indicating the CQ gambicin is inactive. It
may be hypothesized that the inactive CQ gambicin might result from its cleavage by
protease. In fact, the An. gambiae gambicin consists of four disulfide bonds [12], which
should be similar to CQ gambicin. The improper folding of CQ gambicin caused by the
incorrect disulfide bond formation during translation process might affect to its anti-microbial
function. To address these problems, further studies required to recover the active CQ
gambicin will aim to (i) adding the protease inhibitor such as casamino acid in BMMY
culture medium [15], (ii) refolding approach to obtain the correct 3-D protein structure and
(iii) expression in the baculovirus-insect system.
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Acknowledgements
This project was financially supported by the Faculty of Tropical Medicine (Mahidol
University). P.S. received the DAAD (Deutscher Akademischer Austausch Dienst)
scholarship by the German Academic Exchange Service, and Young Scientist Award 2013 by
Merck Millipore Bioscience (Thailand). Additionally, the authors are grateful to Dr. Sungsit
Sungvornyothin from the Department of Medical Entomology, Faculty of Tropical Medicine,
Mahidol University for providing of mosquito samples. The authors also thank for the
assistance of Dr. Amornrat Aroonual, Assist. Prof. Dr. Yuvadee Mahakunkijcharoen and Dr.
Onrapak Reamtong.