Application of DsbA Signal Peptide for Soluble Expression of Leishmania infantum P4 Nuclease in E. coli
Drugs available for treatment of Visceral Leishmaniasis (VL) are toxic and drug resistance is increasing in many parts of the world, thus it seems that vaccine development is an ideal method for prevention and control of VL. P4 nuclease of L. infantum, an amastigote stage specific protein, is considered as a good candidate for VL. Previous efforts for recombinant expression of this protein in E. coli resulted in production as inclusion body form. In the present study, the effect of DsbA signal peptide on periplasmic expression and production of soluble recombinant P4 antigen were examined. DNA extracted from L. infantum was used for amplification of P4 nuclease gene (Li-P4) by PCR. The product was cloned, sequenced and expressed in E. coli under signal sequence DsbA. The results indicated that periplasmic expression of Li-P4 gene in E. coli leads to production of high levels of recombinant protein in soluble form.
Received: August 03, 2011;
Accepted: December 05, 2011;
Published: January 19, 2012
Protozoan parasites of the genus Leishmania causes a spectrum of clinical
disease, including cutaneous, mucocutaneous and Visceral Leishmaniasis (VL).
Approximately 12 million people are infected worldwide with 1.5-2 million new
cases occurring each year (Desjeux, 2004). Leishmania
parasites are dimorphic organisms which exist as promastigote in extracellular
stage and in the sandfly midgut and as amastigote that lives intracellularly
in the phagolysosomes of macrophages in the mammalian host cells (Campos-Neto
et al., 2001; Campbell et al., 2003).
Unfortunately, currently available treatment regimens are non-selective drugs
with significant toxicity and limited efficacy (Jackson
et al., 1990; Grogl et al., 1992).
In the other hands, drug resistance is increasing in many parts of the world
(Croft and Coombs, 2003), thus it is believed that vaccine
development is an ideal method for prevention and control of Leishmania
patients. P4 nuclease is an intracellular amastigote-specific protein that is
initially identified in L. amazonensis (Soong et
al., 1995) and then characterized in L. pifanoi (Kar
et al., 2000), L. major (Farajnia et
al., 2004) and L. infantum (Farajnia et
al., 2011). Immunoblotting analysis of the purified Li-P4 with sera
of VL patients indicated that Li-P4 protein is a highly immunogenic protein
expressed in the amastigote-stage of L. infantum and could be considered
as a potent vaccine candidate against VL caused by L. infantum (Farajnia
et al., 2011). It has been reported that cytoplasmic expression of
Li-P4 in E. coli resulted in accumulation as inclusion body due to disulfide
bonded hydrophobic nature of the protein (Farajnia et
al., 2004). Disulfide bonds are crucial for the folding, stability and
function of many extra-cytoplasmic proteins (Gilbert, 1997;
Ritz and Beckwith, 2001). One of the methods used for
production of soluble proteins is exportation of the protein to the periplasmic
space (Schierle et al., 2003), where correct
disulfide bonds are formed. This translocation is mediated by signal peptides
that carried in the N-terminal of secretary proteins.
The objective of the present study was the evaluation of DsbA Signal peptide on soluble expression of Li-p4 protein in E. coli.
MATERIALS AND METHODS
Parasite and DNA extraction: In this study, Iranian strain of L. infantum was used. Promastigotes were cultured at 26°C in RPMI 1640 medium with glutamine (Gibco BRL) supplemented with 10% heat-inactivated fetal calf serum (Sigma-Aldrich). Organisms were harvested in logarithmic phase and washed with phosphate buffer saline (PBS, pH 7.2). Parasites were disrupted in lysis buffer (50 mM NaCl, 50 mM EDTA, 1% SDS, 50 mM Tris-HCl, pH 8.0) and incubated overnight with proteinase K (100 mg mL-1, Sigma-Aldrich) at 37°C. DNA was then purified by phenol-chloroform extraction and ethanol precipitation.
PCR amplification: A pair of primers was designed based on P4 gene sequence previously reported for Cutaneous Leishmaniasis (CL) strains: Lip4-F:5'-TAGAGCTCGTGGGGCTGCGTGGGT CACAT-3', Lip4-R:5'-ATGTCGACCGCACCTCGCTTCGGACGTG-3'. Each PCR reaction contained 200 ng DNA, 10 p mol each of forward and reverse primers, 1.5 mM MgCl2, 200 μM dNTPs, 1x PCR buffer, 2 unit of Pfu DNA polymerase (Fermentas) and up to 25 μL dH2O. PCR amplification was carried out in 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 63.0°C for 60 sec and extension at 72°C for 60 sec with a final extension cycle at 72°C for 5 min. PCR products were electrophoresed on 1.5% agarose gel and stained by ethidium bromide. The DNA bands were visualized under an ultraviolet light (UV transilluminator) and documented.
Gene cloning: The PCR product was purified by PCR product purification
kit (Roche) and ligated into the pGEM-T easy (Promega) vector. The ligation
reaction was transformed into DH5α (Promega) competent cells and plated
on Luria-Bertani agar (LB Agar) containing ampicillin (50 mg mL-1),
5 bromo-4 chloro-3-indolyl-β-D-galactoside (X-gal: 20 mM) and isopropyl
thio-β-D-galactoside (IPTG: 200 mg mL-1). The white colonies
containing recombinant plasmid were selected (Bothwell et
al., 1990) for plasmid extraction and PCR screening (Feliciello
and Chinali, 1993). Then cloning was verified by restriction digestion and
Expression and solubility analysis: The pGEM-T easy vector containing Li-P4 gene was digested with SalI and Sac I and the insert was purified, subcloned into the SalI-SacI digested pAES30 (Athena system) expression vector and transformed into the E. coli DH5α. The bacteria containing pAES30 Li-P4 was cultured in LB broth medium and grown until OD = 0.5. Expression of recombinant Li-P4 was induced by addition of 1 mM isopropyl-β-D-thiogalactoside (IPTG) then incubated for further 4 h at 37°C and analyzed by sodium dodecyl sulfate-poly acryl amide gel electrophoresis (SDS-PAGE).
For solubility analysis of recombinant Li-P4, E. coli DH5α containing expression vector was cultured in 1 L LB broth medium and following induction with IPTG, the cells was collected by centrifugation (10000 rpm for 15 min). The bacterial sediment was then disrupted in 10 mL lysis buffer (pH = 8, 50 mM NaH2PO4, 300 mM NaCl) by sonication (45 sec pulses interrupted with cooling on ice). The soluble and insoluble fraction was separated by centrifugation of cell sonicate at 12,000 g for 15 min at 4°C and analyzed by SDS-PAGE.
SDS-PAGE analysis: SDS-PAGE was performed using the Laemmli buffer system
(Laemmli, 1970). Prior to electrophoresis, the samples
(cell lysates and fractions) were heated at 100°C for 10 min in dissociating
buffer containing 2% SDS and 5% 2-mercaptoethanol and separated by a 15% SDS-PAGE
and stained by coomassie blue G-250. Protein markers used were phosphorylase
(97.4 kDa), bovine serum albumin (66.3 kDa), ovalbumin (45.0 kDa), lactate dehydrigenae
35.0 kDa), RE Bsp981 (25.0), beta-lactoglobolin (18.4) and lysozyme (14.4 kDa).
Gene cloning: After culture, the L. infantum was subjected to DNA extraction and Li-P4 gene amplification. PCR amplification of P4 nuclease gene from L. infantum resulted in a 862 bp PCR product that was in expected size (Fig. 1). The PCR product was cloned into the pGEM-T easy vector using T-A cloning method and confirmed by restriction digestion (Fig. 2).
Expression and purification of recombinant P4 nuclease: For expression of recombinant Li-P4, the PCR product was subcloned in the SalI-SacI site of pAES30 (Athena) and transformed into the E. coli DH5α (Fig. 3). Induction of recombinant protein expression by IPTG resulted in high level of expression that appeared as a 33 kDa band in SDS-PAGE analysis of cell lysates (Fig. 4).
Solubility analysis of E. coli expressed Li-P4: The cells harboring
pAES30-Li-P4 vector were cultured in 1 L volume and induced by IPTG induction.
The cells were sonicated and sup and pellet fractions were separated by centrifugation.
||Amplification of P4 gene of L. infantum. Lane 1 and
Lane 2: 862 bp Li-P4 gene PCR product, Lane 3: No DNA and Lane 4: 1 kb DNA
||Cloning of Li-P4 gene in pGEM-T vector. Lane 1: Undigested
recombinant plasmid, Lane 2: SalI-SacI digested recombinant plasmid and
Lane 3: 1 kb DNA ladder
||Cloning Li-P4 gene in pAES30 vector. Lane 1: Recombinant plasmid,
undigested, Lane 2: SalI-SacI digested recombinant plasmid and Lane 3: 1
kb DNA ladder
||SDS-PAGE analysis of recombinant Li-P4 produced in E.coli
DH5 , Lane 1: Bacterial lysate before induction, Lane 2: Bacterial lysate1
h after induction with IPTG, Lane 3: Bacterial lysate 2 h after induction,
Lane 4: Bacterial lysate 3 h after induction and Lane 5: Molecular weight
||Solubility analysis of recombinant Li-P4 produced in E.coli
DH5α, Lane 1: Bacterial lysate before induction, Lane 2: Bacterial
lysate after induction with IPTG, Lane 3: Cells pellet 3 h after induction
with IPTG, Lane 4: Cells soup 3 h after induction, Lane 5: Cells soup 24
h after induction, Lane 6: Cells pellet 24 h after induction and Lane 7:
Molecular weight marker
SDS-PAGE analysis of fractions after induction for different hours revealed
that Li-P4 was present mainly in soluble fraction after induction for 3 h whereas,
expression for longer times increased protein in insoluble fraction with complete
insoluble expression in 24 h samples (Fig. 5).
Different vaccine candidates have been tried for prevention of VL among them
P4 nuclease antigen showed promising results in several studies. P4 protein
is a single strand specific nuclease that has shown to be conserved in different
species of Leishmania (Soong et al., 1995;
Kar et al., 2000; Farajnia
et al., 2004). We recently characterized P4 nuclease in L. infantum
and showed that sera from VL patients highly reacted with this protein indicating
immunogenic nature of this protein (Farajnia et al.,
2011). These finding suggested Li-P4 as a promising vaccine candidate for
Previous studies have shown that Li-P4 appears as inclusion body during recombinant
expression in E. coli. Structural analysis shows that Li-P4 is a highly
hydrophobic protein containing 2 disulfide bonds. Formation of disulfide bonds
is critical for correct folding, stability and export of many secreted proteins
by gram-negative bacteria (Missiakas and Raina, 1997;
Rietsch and Beckwith, 1998). One of the methods for
production of recombinant proteins in soluble form in the bacteria is exportation
of proteins to the periplasmic space (Schierle et al.,
2003). Translocation to the periplasm of E. coli has several advantages
over cytoplasmic expression including simplified downstream processing, higher
product solubility, enhanced biological activity and N-terminal authenticity
of the expressed proteins (Cornelis, 2000; Macrides,
1996; Mergulhao et al., 2004). Furthermore,
for proteins expressed in the periplasmic space, a simple osmotic shock or cell
wall permeabilization can be used to obtain the product without contamination
with cytoplasmic proteins (Mergulhao et al., 2004;
Shokri et al., 2003).
Different signal peptides have been attempted for periplasmic expression of
proteins, including OmpA (Chen et al., 1980),
DsbA (Schierle et al., 2003), PelB (Guo
et al., 1995) and PhoA (Inouye et al.,
1982), among them DsbA signal peptide has shown good results in several
studies (Schierle et al., 2003; Soares
et al., 2003). DsbA is an oxidoreductase enzyme that are exported
from the cytoplasm to the periplasmic space by its signal peptide (Jonda
et al., 1999; Randall et al., 1998).
Schierle et al. (2003) has shown that in comparison
to PhoA, DsbA signal sequence exports the cytoplasmic protein thioredoxin 1
efficiently to the periplasmic space whereas, translocation by PhoA signal sequence
had very low yields (Debarbieux and Beckwith, 1998).
It has been suggested that the reason for this effect is related to the ability
of DsbA signal sequence to direct the fused protein into the co-translational
SRP pathway (Schierle et al., 2003). It has also
shown that recombinant expression of human growth hormone by using DsbA signal
peptides leads to higher expression level and solubility compared to expression
without DsbA signal (Soares et al., 2003). In
the present study we used the DsbA signal peptide for periplasmic soluble expression
of Li-P4 in E. coli. The results showed that recombinant Li-P4 highly
expressed using this signal peptide. This finding is consistent with previous
reports about the potential of DsbA signal peptide (Schierle
et al., 2003; Debarbieux and Beckwith, 1998).
Analysis of time course of expression revealed that the recombinant protein
appeared in soluble form 3 h after induction with IPTG whereas, continuation
of incubation to 6 h and more result in gradual formation of inclusion body.
The reason for such phenomena is not clear and may be related to the limited
periplasmic export capacity of E. coli. By accumulation of recombinant
protein expressed in cytoplasm of the cells, the proteins aggregate gradually
results in inclusion body formation.
In conclusion, the results of present study indicated that expression of hydrophobic Li-P4 under DsbA signal peptide leads to soluble expression in E. coli. This finding could be exploited for soluble expression of other recombinant proteins that are expressed as inclusion body in bacteria.
1: Bothwell, A.L., G.D. Yancopulos and F.W. Alt, 1990. Methods for Cloning and Analysis of Eukaryotic Genes. Jones and Bartlett Publishers, Boston, pp: 247-260.
2: Campbell, K., H. Diao, J. Ji and L. Soong, 2003. DNA Immunization with the Gene Encoding P4 Nuclease of Leishmania amazonensis Protects Mice against Cutaneous Leishmaniasis. J. Infect., 71: 6270-6278.
3: Campos-Neto, A., R. Porrozzi, K. Greeson, R.N. Coler and J.R. Webb et al., 2001. Protection against Cutaneous Leishmaniasis induced by recombinant antigens in Murine and Nonhuman Primate models of the human diseas. J. Infect., 69: 4103-4108.
4: Chen, R., W. Schmidmayr, C. Kramer, U. Chen-Schmeisser and U. Henning, 1980. Primary structure of major outer membrane protein II (OmpA protein) of Escherichia coli K-12. Proc. Natl. Acad. Sci., 77: 4592-4596.
5: Croft, S.L. and G.H. Coombs, 2003. Leishmaniasis-current chemotherapy and recent advances in the search for novel drugs. Trends. Parasitol., 19: 502-508.
PubMed | Direct Link |
6: Cornelis, P., 2000. Expressing genes in different Escherichia coli compartments. Curr. Opin. Biotechnol., 11: 450-454.
CrossRef | PubMed | Direct Link |
7: Debarbieux, L. and J. Beckwith, 1998. The reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc. Natl. Acad. Sci. USA., 95: 10751-10756.
8: Desjeux, P., 2004. Leishmaniasis: Current situation and newperspectives. Comp. Immunol. Microbiol. Infect. Dis., 27: 305-318.
9: Farajnia, S., L. Rahbarnia, B.M. zanjani, M.H. Alimohammadian and S.A. Oskoee et al., 2011. Molecular cloning and characterization of P4 nuclease fromLeishmania infantum. Enzyme Res., 2011: 6-6.
CrossRef | Direct Link |
10: Farajnia, S., M.H. Alimohammadian, N.E. Reiner, M. Karimi, S. Ajdari and F. Mahboudi, 2004. Molecular characterization of a novel amastigote stage specific class I nuclease from Leishmania major. Int. J. Parasitoogyl., 34: 899-908.
11: Feliciello, I. and G. Chinali, 1993. A modified alkaline lysis method for the preparation of highly purified plasmid DNA from Escherichia coli. Anal. Biochem., 212: 394-401.
Direct Link |
12: Gilbert, H.F., 1997. Protein disulfide isomerase and assisted protein folding. J. Biol. Chem., 272: 29399-29402.
13: Grogl, M., T.N. Thomason and E.D. Franke, 1992. Drug resistance in leishmaniasis: its implication in systemic chemotherapy of cutaneous and mucocutaneous disease. Am. J. Trop. Med. Hyg., 47: 117-126.
14: Guo, W., L. Gonzalez-Candelas and P.E. Kolattukudy, 1995. Cloning of a novel constitutively expressed pectate lyase gene pelB from Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) and characterization of the gene product expressed in Pichia pastoris. J. Biol. Chem., 177: 7070-7077.
Direct Link |
15: Inouye. H., W. Barnes and J. Beckwith, 1982. Signal Sequence of Alkaline Phosphatase of Escherichia coli. J. Biol Chem., 149: 434-439.
16: Jackson, J.E., J.D. Tally, W.Y. Ellis, Y.B. Mebrahtu and P.G. Lawyer et al., 1990. Quantitative in vitro drug potency and drug susceptibility evaluation of Leishmania ssp. from patients unresponsive to pentavalent antimony therapy. Am. J. Trop. Med. Hyg., 43: 464-480.
Direct Link |
17: Jonda, S., M. Huber-Wunderlich, R. Glockshuber and E. Mossner, 1999. Complementation of DsbA deficiency with secreted thioredoxin variants reveals the crucial role of an efficient dithiol oxidant for catalyzed protein folding in the bacterial periplasm. EMBO. J., 18: 3271-3281.
18: Kar, S., L. Soong, M. Colmenares, K. Goldsmith-Pestana and D. McMahon- Pratt, 2000. The immunologically protective P-4 antigen of Leishmania amastigotes. A developmentally regulated single strand-specific nuclease associated with the endoplasmic reticulum. J. Biol. Chem., 275: 37789-37797.
19: Makrides, S.C., 1996. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev., 60: 512-538.
Direct Link |
20: Mergulhao, F.J.M., G.A. Monteiro, J.M.S. Cabral and M.A. Taipa, 2004. Design of bacterial vector systems for the production of recombinant proteins in Escherichia coli. J. Microbiol. Biotechnol., 14: 1-14.
21: Missiakas, D. and S. Raina, 1997. Protein misfolding in the cell envelope of Escherichia col.: New signaling pathways. Trends. Biochem. Sci., 22: 59-63.
22: Randall, L.L., T.B. Topping, V.F. Smith, D.L. Diamond and S.J. Hardy, 1998. SecB: A chaperone fro Escherichia coli. Methods. Enzymol., 290: 444-459.
23: Rietsch, A. and J. Beckwith, 1998. The genetics of disulfide bond metabolism. Annu. Rev. Genet., 32: 163-184.
24: Ritz, D. and J. Beckwith, 2001. Roles of thiol-redox pathways in bacteria. Ann. Rev. Microbiol., 55: 21-48.
25: Schierle, C.F., M. Berkmen, D. Huber, C. Kumamoto, D. Boyd and J. Beckwith, 2003. The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway. J. Bacteriol., 185: 5706-5713.
26: Shokri, A., A.M. Sanden and G. Larsson, 2003. Cell and process design for targeting of recombinant protein into the culture medium of Escherichia colim. Appl. Microbiol. Biotechnol., 60: 654-664.
27: Soares, C.R.J., F.I.C. Gomide, E.K.M. Ueda and P. Bartolin, 2003. Periplasmic expression of human growth hormone via plasmid vectors containing the lPL promoter: Use of HPLC for product quantification. Pr. Eng., 16: 1131-1138.
Direct Link |
28: Soong, L., S.M. Duboise, P. Kima and D. Mcmahon-Pratt, 1995. Leishmania pifanoi amastigote antigens protect mice against cutaneous leishmaniasis. Infect. Immun., 63: 3559-3566.
Direct Link |
29: Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.
CrossRef | Direct Link |