Journal of Genetic Engineering and Biotechnology (2014) xxx, xxx–xxx
Academy of Scientific Research & Technology and National Research Center, Egypt
Journal of Genetic Engineering and Biotechnology www.elsevier.com/locate/jgeb
ARTICLE
Cloning and expression of FimA-c3d recombinant protein Hassan Hussein Musa a,b, Weijuan Zhang a, Jie Tao a, Yuankun Guan Xiaoli Duan a,c, Yang Yang a,c, Chunhong Zhu a,d, Huifang Li d, Guoqiang Zhu a,c,*
a,c
,
a
College of Veterinary Medicine, Yangzhou University, China Faculty of Medical Laboratory Sciences, University of Khartoum, Sudan c Ministry of Education Key Lab for Avian Preventive Medicine, China d Jiangsu Institute of Poultry Science, Yangzhou 225003, China b
Received 12 October 2012; revised 19 December 2012; accepted 30 March 2014
KEYWORDS FimA-c3d; Recombinant protein
Abstract Recombinant protein consisting of an antigen fused to C3d may elicit a more robust immune response than the antigen alone. The objective of the present study was to clone FimAc3d recombinant DNA into pCold-TF vector and successfully express in prokaryotic expression vector. FimA subunit of type I fimbriae from Salmonella enterica serovar Enteritidis was conjugated to mouse complement fragment molecule C3d. FimA and FimA-mC3d, FimA-mC3d2 and FimAmC3d3 were cloned into the expression vector pCold-TF. Constructions of the recombinants pCold-TF-fimA with different copies of C3d were confirmed by digestion with restriction enzymes and sequencing. Soluble fusion proteins of FimA with different copies of C3d were induced by IPTG and were expressed in Escherichia coli BL21 (DE3) under optimal conditions. The results showed that the proteins induced from recombinants pCold-TF-fimA, pCold-TF-fimA-mC3d, pCold-TF-fimA-mC3d2 and pCold-TF-fimA-mC3d3 were 70, 100, 130 and 160 kDa, respectively. The fusion protein was recognized by rabbit anti-fimbriae polyclonal antibodies, and then visualized by goat anti-rabbit polyclonal antibodies with a chrome appearance by enzyme-subtract interaction. The recombinant proteins were separately purified by Ni-TED (tris-carboxymethyl ethylene diamine) immobilized metal iron affinity chromatography (IMAC). Finally we conclude that antigen conjugated with mC3d can be functionally expressed in prokaryotic vectors. ª 2014 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology.
* Corresponding author at: College of Veterinary Medicine, Yangzhou University, China. E-mail addresses:
[email protected],
[email protected] (G. Zhu). Peer review under responsibility of National Research Center, Egypt.
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1. Introduction Fimbriae of Salmonella enterica serovar Enteritidis are used for colonization and invasion into host cells, and have drawn considerable interest because fimbriae can serve as potential immunogens against many pathogenic bacteria that colonize epithelial surfaces [24]. The main fimbriae of the S. enterica
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serovar Enteritidis are SEF14, SEF17 and SEF21 composed of SefA, AgfA and FimA fimbrial proteins, respectively [13]. Fimbria-based vaccines are hypothesized to protect the host against the adherence of pathogens by blocking the attachment of organisms to the intestinal mucosa [31]. Recently, scientist’s interest has revolved around the effecter molecules generated by the innate response and their role in shaping acquired immunity [6]. Recombinant protein consisting of an antigen fused to C3d may elicit a more robust immune response than the antigen alone. Dempsey et al. [2] demonstrated that a recombinant protein containing three copies of C3d attached to the carboxy terminus of hen egg lysozyme (HEL) could elicit a primary immune response at a concentration 10,000-fold more than that required for the unmodified HEL protein. A major bottleneck in structural biology is that many human proteins are expressed very poorly or are expressed well but not in soluble forms [14]. These difficulties may arise due to the improper folding of human proteins in Escherichia coli cells, where they are quickly digested by proteases or accumulated as inclusion bodies [14]. A number of expression systems have been developed to overcome this problem, for example, by coexpressing human proteins with molecular chaperones [21], expressing them as fusions with maltose-binding protein [4], glutathione S-transferase [27], or expressing them at low temperatures using cold shock vectors [23]. Heat-shock response from bacteria to humans has been extensively studied, while cold-shock response has caught the attention of researchers relatively recently [22]. A major reason why heat shock is extensively studied is because it causes well-defined damage to the cells, i.e. unfolding or denaturation of proteins. In contrast, cold shock does not cause such well-defined cellular damage. Cold-shock response is classically exhibited when an exponentially growing culture is shifted from its optimum growth temperature to a lower temperature [22]. Several cold shock proteins have been detected not only in E. coli but also in many other bacteria [17,30,25,10]. Certain aspects of bacterial coldshock response such as regulation of expression of CspA homologues have been extensively reviewed [5,9,29,35]. CspA function was proposed to be an RNA chaperone [35,8]. The CspA family is essential for E. coli cells to adapt to low temperature [32,3]. A peptidyl prolyl isomerase (trigger factor-TF), that catalyzes the cis/trans isomerization of peptide bonds N-terminal to the proline residue was identified in E. coli. This enzyme is induced upon cold shock at a modest level after a growth lag period of 2–3 h, its overexpression leads to enhanced viability [12]. The objective of the present study was to clone FimAc3d recombinant DNA into pCold-TF vector and successfully express in the prokaryotic expression system.
2.2. Reagents Restriction enzymes NdeI, BamHI, BglII and T4 DNA ligase were purchased from NEB’s, Amp (Ampicillin) and Expand High Fidelity PLUS PCR System was purchased from the Roche Company, Purification of Polyhistidine-Tagged Proteins (Proteins Ni-TED2000) were purchased from MACHEREY-NAGE company, DNA marker and lysozyme were purchased from the Takara Company, High molecular weight marker and Pre-stained low molecular weight marker were purchased from the Fermentas Company, 3,30 -diaminobenzidine (3,30 -diamino benzidine, DAB) and agarose gel DNA extraction kit (centrifugal columnar) were purchased from the BBI (Bio basic Inc.) company, Isopropyl-b-D-thiogalactoside (IPTG) were purchased from Takara Biotechnology Co., Ltd., goat anti-rabbit HRP-IgG antibodies were purchased from the Boster Company, sodium dodecyl sulfate (SDS), ammonium persulfate (APS), N,N,N,N-tetramethylethylenediamine (TEMED) is from the AMRESCO company, electrophoresis grade acryl amide and Tris-base were from BioSharp, and N,N-methylene-bis-acrylamide was from Gibco. 2.3. Bacteria growth and DNA extraction
2. Materials and methods
Salmonella enterica serovar Enteritidis was cultured over night at 37 C with vigorous agitation and then DNA was extracted. FimA gene (6949132) was amplified by an upper primer with NdeI restriction enzyme site: 5’-CGC CATATG AAA CAT AAA TTA ATG ACC TCT A-3’ and lower primer with NotI and BamHI restriction enzymes site: 5’-TCG GCG GCC GCG GAT CCT TCG TAT TTC ATG ATA AAG GTG-3’. PCR was carried out in a total volume of 25 ll containing 2.5 ll DNA template, 1.5 ll of 2.5 mM dNTP mixture, 2.5 ll of 10 · PCR buffer, 1 ll upper primer, 0.5 ll lower primer, 0.5 ll Ex Taq polymerase and 16.5 ll sterilized distilled water. PCR conditions were initial denaturation at 94 C for 4 min, followed by 30 cycles at 94 C for 1 min, annealing at 56 C for 1 min and extended at 72 C for 1 min, and finally the PCR product was extended at 72 C for 10 min. The 550 bp of FimA gene was excised from the gel and purified using DNA purification kit. Purified product was then cloned into pMD18-T simple vector and transformed into E. coli DH5a competent cells. The E. coli DH5a carrying recombinant plasmid (pMD18-T + FimAgene) was grown on LB plates containing 100 lg/ml ampicillin at 37 C overnight, and the single clone was grown in 5 ml LB broth plus ampicillin (100 lg/ml) at 37 C to OD600 of 0.4–0.5. Recombinant plasmids were extracted and digested by restriction enzyme and sequenced.
2.1. Strains and vectors
2.4. Construction of recombinant protein
Standard strains of Salmoenlla enterica serovar Enteritidis SD-2, E. coli DH5a and recombinant protein expression recipient strain E. coli BL21 (DE3), PCold-TF expression vector and recombinant plasmid pUC-mC3d, pUC-mC3d2 and pUC-mC3d3 and Salmonella Enteritidis -type pilus polyclonal (rabbit) were preserved in our laboratory. Primers were synthesized by Shanghai Gene Core Biotechnology Co., Ltd., and pMD18-T. Simple vector was purchased from Takara Biotechnology Co., Ltd.
Recombinant plasmids pMD18-T-FimA and pCold-TF vectors were digested with NdeI and BamHI restriction enzymes. Both fimA gene and pCold-TF expression vector were recovered from the gel using the agarose gel DNA purification kit. Purified fimA DNA was cloned into pCold-TF expression vector (Fig. 1). The recombinant pCold-TF-fimA was transformed into the E. coli DH5a competent cells; positive clones were selected and determined by restriction enzyme NdeI and BamHI. Clones of
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Journal of Genetic Engineering and Biotechnology (2014), http://dx.doi.org/10.1016/
Cloning and expression of FimA-C3d recombinant protein
Figure 1
pCold TF DNA plasmid map.
the pUC-C3dn plasmid generated according to strategy of Dempsey et al. [2] were gifted from Drs. Leonard J Bello and Lingshu Wang, University of Pennsylvania, USA. Each C3d was connected with a linker sequence encoding glycine/serine repeats GS (G4S)2 GS, of which potential proteolytic cleavage sites were mutated by BamH1 and BglII fusion to mutate an arginine to a glycine. The plasmids were digested with BamHI and BglII to release the BLc3d, BLc3d2 and BLc3d3 fragments, and pCold-TF-fimA clone was digested with BamHI. The digested products were recovered from the gel using the agarose gel DNA purification kit, then the BLc3d, BLc3d2 and BLc3d3 fragments were respectively cloned into the pCold-TF-fimA. The recombinant DNA was transformed into the E. coli DH5a competent cells and the positive clones were selected and determined by PCR and restriction enzyme analysis. 2.5. Recombinant protein expression Two microliters of each of pCold-TF-fimA, pCold-TF-fimABLc3d, pCold-TF-fimA-BLc3d2 and pCold-TF-fimA-BLc3d3 plasmid was used to transform 200 ll E. coli BL21 (DE3). The E. coli BL21 (DE3) carrying pCold-TF-fimA-BLc3d was grown on LB plates containing 100 lg/ml ampicillin at 37 C overnight. Then the single clone was grown in 5 ml LB broth plus ampicillin (50 lg/ml) at 37 C to OD600 of 0.4–0.5. After standing for 30 min at 15 C, the culture was split into two aliquots. One was induced by IPTG to a final concentration of 0.1 mmol/L at 15 C for 24 h and the other un-induced was considered as control. Cells harvested were lysed by sonication and subjected to 8% SDS–PAGE. The protein on 8% SDS– PAGE was transferred to nitrocellulose membrane (0.35 mA/ cm2).The membrane was blocked with 10% nonfat dry milk and incubated with a primary antibody (Type 1 fimbriae polyclonal (Rabbit) antibody) (1:100) and with a secondary antibody (Goat Anti-Rabbit IgG) (1:1500). The recombinant proteins were separately purified by Ni-TED (tris-carboxymethyl ethylene diamine) immobilized metal iron affinity chromatography (IMAC). 3. Results and discussion Molecular adjuvant C3d can be linked to different antigens, and effectively enhances the titer and life-span of specific
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3 antibodies against these antigens [20,15]. Comparison of the C3d sequences of human (hC3d), mouse (mC3d) and bovine (boC3d) showed 84.1% amino acid homology between hC3d and mC3d and 80.5% homology between hC3d and boC3d, they either showed the function of immune adjuvant in the mammalian model [37]. Certain proteins are not produced efficiently at 37 C, while they can be overproduced in large quantities at low temperatures by using cold-inducible promoters, for example, the promoter of cspA, encoding the major cold shock protein of E. coli [22]. In the present study soluble Fim A protein fused to C3d was expressed in prokaryote expression vector. The fimA gene was amplified by PCR using a pair of primers and the DNA template from Salmonella enterica serovar Enteritidis standard strain SD-2 genomic DNA. FimA gene was digested and cloned into pCold-TF expression vector to construct recombinant plasmid pCold-fimA, which was confirmed by restriction enzymes and sequencing. The different copies of mC3d, mC3d2 and mC3d3 were obtained through the digestion of recombinant plasmids pUC-mC3d, pUC-mC3d2 and pUC-mC3d3, and were cloned into the recombinant plasmid pCold-fimA to construct the recombinant plasmids, i.e. pCold-fimA-mC3d, pCold-fimA-mC3d2 and pCold-fimA-mC3d3. The fusion proteins were optimally induced by IPTG at 15 C for 24 h, and were expressed in E. coli BL21 (DE3). A variety of methods have been developed for including protein production at low temperature, and for fusion protein expression using soluble protein tags [11]. When an E. coli culture growing at 37 C is shifted to low temperature, cell growth stops temporarily. During this growth lag period, called the acclimation phase, the synthesis of most cellular proteins is sharply reduced, whereas a specific set of proteins termed ‘‘cold shock proteins’’ are induced [35,33]. Among these cold shock-inducible proteins, CspA is the major cold shock protein in E. coli [7], which can be induced to a level of 10 · 106 molecules/cell [1,34]. CspA has been implicated to be a negative regulator of its own gene expression, because in a strain with a deletion mutation of the cspA coding sequence, the amount of the truncated cspA mRNA was much more than that in its parental strain, and the production of such truncated cspA mRNA was prolonged in the mutant cells [36]. The pCold vector used in the present study contains the promoter of the cold shock-inducible gene for the major cold shock protein CspA and the gene’s 134-base 5 untranslated region, allowing a very high level of expression of a cloned gene upon cold shock [23]. This vector also contains a lac operator downstream of the cspA promoter so that the expression of a cloned gene can be tightly regulated not only by temperature but also by lac inducers such as isopropyl-b-D-1-thiogalactopyranoside (IPTG) [14]. TF in pCold vector has a ‘maintenance and repair’ function as it helps protein synthesis and folding to continue at low temperature [12]. It accelerates proline-limited steps in protein folding with a very high efficiency. In addition, it associates with ribosomes and influences the folding of newly formed protein chains [19]. Although the pCold vector is designed for the production of proteins at 15 C, the CspA promoter is also active at 37 C and some proteins can be produced in high yields at room temperature with the pCold vector [14]. The expressed proteins with the molecular weight of 70 kDa, 100 kDa, 130 kDa and 160 kDa, respectively, were confirmed by SDS–PAGE (Fig. 2). The antigenicity of the
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recombinant proteins expressing FimA was demonstrated by Western blotting with the goat against rabbit polyclonal antibody of type I fimbriae (Fig. 3). The recombinant proteins were separately purified by Ni-TED (tris-carboxymethyl ethylene diamine) immobilized metal iron affinity chromatography (IMAC) (Fig. 4). Kobayashi et al. [14] demonstrated that the expression and solubility levels of a number of human proteins are significantly improved when these proteins are expressed as fusions with the NTDs of protein S by using pCold vector [14]. The pCold-GST expression system was applied to express 10 proteins that could not be expressed using conventional E. coli expression method, and nine of these proteins were successfully obtained in the soluble fraction [11]. Hayashi and Kojima [11] suggested that the pCold-GST expression system
Figure 4 SDS–PAGE of purified recombinant proteins (1) FimA; (2) FimA-mC3d; (3) FimA-mC3d2; (4) FimA-mC3d3; and M. High (Molecular weight range: 44.3–200 kDa).
Figure 2 SDS–PAGE for recombinant proteins (1) FimA; (2) Control vector; (3) FimA-mC3d3; (4) FimA-mC3d2; (5) FimAmC3d; and M. Low (Molecular weight range: 14.3–97.2 kDa).
can be utilized to improve the expression and purification of various proteins [11]. This new technology can also be used for the analysis of protein–protein interactions and for the structural study of proteins which are difficult to express or are insoluble [14]. C3d could enhance antibody responses directed toward a specific antigen encoded by a DNA vaccine [26]. Three copies of murine C3d could dramatically enhance antibody responses to a specific antigen, being 100-fold more effective than incomplete Freunds adjuvant [2,18]. Wang [28] reported that the bovine homologue of C3d (boC3d) coupled to the E2 envelope protein of bovine viral diarrhea virus greatly enhanced immunogenicity in mice. Similarly, chicken C3d-P29 linked to the F gene of Newcastle disease virus (NDV) enhanced immunogenicity in chickens [16]. Therefore, in the present study we conclude that antigen conjugated with mC3d can be functionally expressed in prokaryotic vectors. Acknowledgements The authors are grateful to Drs. Leonard J Bello and Lingshu Wang, University of Pennsylvania, for providing recombinant pUC-mC3d, pUC-mC3d2 and pUC-mC3d3 plasmids. This study was supported by Grants from the Chinese National Science Foundation Grants No. 31072136, 30771603, 30710103069, 31101826, Genetically Modified Organisms Technology Major Project of P. R. China (2009ZX08006004B), the Jiangsu High Education Key Basic Science Foundation (08KJA230002), Ministry of Agriculture, P. R. China Grant (No. 200803020), Program for Changjiang Scholars and Innovative Research Team in University ‘‘PCSIRT’’ (IRT0978). References
Figure 3 Western blotting of the recombinant proteins (1) FimA; (2) FimA-mC3d; (3) FimA-mC3d2; (4) FimA-mC3d3; and M. Pre-stained Low (Molecular weight range: 14.3–97.2 kDa).
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