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Vectors for enhanced gene expression and protein purification in Salmonella
Gene 421 (2008) 95–98
Contents lists available at ScienceDirect
Gene j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g e n e
Methods paper
Vectors for enhanced gene expression and protein purification in Salmonella Mohamed N. Seleem a,c, Mohammed Ali b, Stephen M. Boyle c, Nammalwar Sriranganathan c,⁎ a
Institute for Critical Technology and Applied Science, Virginia Polytechnic Institute and State University, USA Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Assiut University, Egypt c Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, USA b
A R T I C L E
I N F O
Article history: Received 28 October 2007 Received in revised form 31 January 2008 Accepted 5 June 2008 Available online 22 June 2008
A B S T R A C T Improved expression vectors have been tested for protein expression studies in Salmonella spp. They are derived from the broad host range expression vector pNSGroE [Seleem, M.N., Vemulapalli, R., Boyle, S.M., Schurig, G.G. and Sriranganathan, N., 2004. Improved expression vector for Brucella species. Biotechniques 37, 740–744] and have several advantages (i) small in size (less than 3 kb); (ii) possess eleven unique restriction site to facilitate directional cloning; (iii) express proteins as His-tagged fusions for easy detection and purification; (iv) carry different promoters for various level of expression and (v) carry an UP element and RNA stem–loop for enhanced gene expression. We have demonstrated the ability of the new vectors to stably express heterologous proteins in Salmonella. We also demonstrated the utility of our vectors by detecting expression and purification of recombinant GFP. Our results suggest that these new vectors should improve gene expression in Salmonella, particularly those aimed at foreign antigen delivery. © 2008 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Several enhanced expression vectors have been constructed for cloning, gene expression, protein purification and promoter selection in O. anthropi (Seleem et al., 2006). The backbone of all vectors originated from the pNSGroE, a Brucella expression vector (Seleem et al., 2004) and a derivative of the pBBR1 originally isolated from Bordetella bronchiseptica (Antoine and Locht, 1992); the ability of the vectors to propagate in a wide variety of gram-negative bacteria encouraged us to test them in Salmonella. These vectors contain various promoters for different levels of expression and also His-tag fusion in the N-terminus to facilitate protein detection and purification. The functionality of all vectors was assessed by cloning the β-galactosidase gene and determining the level of protein expression and LacZ activity. We have exploited the transcriptional activity of the strong regulated coliphage T5 promoter (Wang et al., 2000), and trc hybrid promoter (Amann et al., 1983) in Salmonella. We were able to express, detect and purify recombinant proteins directly from Salmonella. We were able to further enhance gene expression in Salmonella by incorporation of three elements: an UP element, a RNA stem–loop and a tandem tag fusion (Estrem et al., 1998; Paulus et al., 2004). The expression vectors used in the current study proved to be easily transformed into and stably maintained in Salmonella.
2.1. Expression vectors
Abbreviations: CTD, Carboxy-terminal domain; GFP, Green fluorescence protein; LacZ, β-galactosidase gene; MCS, Multiple cloning site; UP, Upstream element. ⁎ Corresponding author. Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1410 Prices Fork Rd, Blacksburg, VA, 24061, USA. Tel.: +1 540 231 7171; fax: +1 540 231 3426. E-mail addresses: [email protected], [email protected] (N. Sriranganathan). 0378-1119/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2008.06.021
The sequences of all vectors, which are reported in this paper have been deposited in the GenBank database. Their description, sizes and accession numbers are listed in Table 1. 2.2. Expression of β-galactosidase (LacZ) In order to study the expression and activity of the cloned promoters inside Salmonella, the promoterless Escherichia coli βgalactosidase (lacZ) gene was amplified from pRSETB/β-gal as described before (Seleem et al., 2004) and cloned into all expression vectors downstream of the various promoters. 2.3. GFP constructs A promoterless Green Fluorescence Protein gene (gfp) was excised from pGFPuv vector (BD Biosciences Clontech) and cloned in-frame and down stream of the promoter in the multiple cloning site (MCS) of pNSTrcD and pNSCh. GFP was used as a visual marker for gene expression, Western blotting and protein purification. 2.4. Competent cells preparation and transformation To prepare Salmonella enterica serotype Choleraesuis (S. Choleraesuis) competent cells, 300 μl of culture (OD600 = 1) was spread on TSA plates, and incubated overnight at 37 °C. The bacteria were gently
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(Vemulapalli et al., 2000) were followed to determine and compare the levels of β-galactosidase expression from different promoters.
Table 1 Descriptions of the plasmids that were constructed and used in this study Plasmid
Size bp
Promoter
Type of expression
Accession number
2.5. Protein purification
pNSCh pNSKan pNSAmp pNST5 pNSTrcD pNSGroE pNSGroE2His pNSChE2His pNSTrcB pNSTrc
2903 2809 2891 2810 2937 2863 2881 2921 2938 2932
Ch r Kanr Ampr Phage T5 trc hybrid + UP B. abortus groE groE + RNA stem loop Ch + RNA stem loop trc hybrid + UP trc hybrid
Constitutive Constitutive Constitutive Regulated Regulated Constitutive Constitutive Constitutive Regulated Regulated
DQ412050 DQ412052 DQ412053 DQ412054 DQ412056 AY576605 DQ412049 DQ412051 DQ412057 DQ412055
The pNSTrcD-gfp expression vector with gfp expressed under the TrcD promoter was transformed into S. Choleraesuis as described earlier. The GFP was purified from recombinant S. Choleraesuis using 2 methods: a) Ni-NTA Spin Columns (Qiagen) under denaturing condition using 8 M urea and according to the manufacturer's instructions, 20% ethanol was added to the washing buffer to reduce contaminant proteins. b) Ni-NTA agarose (Qiagen) and 6 M guanidine HCl. 20 mM imidazol and 20 mM β-mercaptoethanol were added to the lysis buffer and 1% Triton X-100 was added to the washing buffer to reduce contaminating proteins. The concentration of the purified recombinant protein was determined by BCA Protein Assay kit (Pierce) using the manufacturer's enhanced test tube procedure after removal of urea by Microcon centrifugal filter devices YM-10 (Millipore). The purity of the extracted protein was estimated following visualization on SDS-PAGE.
scraped from the plates and washed twice with ice-cold 10% glycerol. The cells were resuspended to a density of approximately 1011 cells/ml in icecold 10% glycerol. The plasmids containing either lacZ or gfp were transformed into S. Choleraesuis by electrotransformation with a Gene Pulser (BTX) set at 2.7 kV, 25 μF and 200 Ω using a 1 mm gap cuvette (Eppendorf). After electroporation, the cells were transferred to SOC media (Invitrogen) and incubated at 37 °C, with shaking at 200 rpm, for 2 h to allow expression of the antibiotic resistance gene. The transformation mixture was plated on several TSA plates containing 30 μg chloramphenicol. Transformation efficiency and stability of the plasmids were calculated as described before (Seleem et al., 2004). Previously described procedures
2.6. Western blot To test the fusion of the His-epitope tagging and efficiency of detection, Western blot analysis was performed by separating the total cell lysates of recombinant S. Choleraesuis expressing GFP and 15 μg of purified recombinant GFP on a 12% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was incubated overnight
Fig. 1. LacZ activity from different promoters in S. Choleraesuis. Promoter activity of each construct was determined and represented as Miller unit. The data are means ± SD of triplicate assays. A, activity of lacZ under different promoter in S. Choleraesuis; B, effect of UP element on the expression of trc promoter; C and D, effect of RNA stem–loop on Ch and groE promoter.
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with a mixture of Anti-HisG-HRP antibody (1:3000) (Invitrogen) and Anti-His6-Peroxidase (Roche) (1:3000). The combination of antibodies that recognize 6×His and one glycine (Anti-HisG-HRP) with antibodies that recognize only 6×His (Anti-His6-Peroxidase) produced more intense band and higher sensitivity of detection. 3. Results and discussion All expression vectors were successfully introduced into S. Choleraesuis with an efficiency of 4.5 × 104 transformants per μg DNA. All plasmids were found to be stably maintained after 8 serial passages over 16 days on TSA plates in the absence of chloramphenicol selection (Seleem et al., 2004). All plasmids constructs were successfully transformed and stably maintained in S. Typhimurium, S. Dublin and S. Newport. 3.1. Promoter activity A comparison was made between the relative levels of LacZ activity expressed under different promoters. All tested promoters were able to express inside S. Choleraesuis with various strengths (Fig. 1A). The chloramphenicol promoter (Ch promoter) was the strongest promoter. In this study, we have exploited the transcriptional activity of the strong coliphage T5 promoter (Wang et al., 2000) and trc hybrid promoter (Amann et al., 1983) in S. Choleraesuis and demonstrated that both promoters are functional (Fig. 1A). The T5 promoter and trc hybrid promoter have the lac operator (lacO), that permits binding of the lac repressor to regulate transcription in bacterial strains that harbor the lacIq gene. Regulated expression using the T5 promoter and trc hybrid promoter in S. Choleraesuis is possible by incorporating the lacIq gene into the plasmid (Chen and Winans, 1991). 3.2. UP element DNA regions that flank a gene's promoter play an important role in determining transcription efficiency by interacting with the carboxyterminal domain (CTD) of RNA polymerase α-subunit (Estrem et al.,1998). We placed an adenine (A)-rich and A+T rich upstream element (UP) (Estrem et al., 1998) between −40 and −59 bp of the core trc promoter
Fig. 3. Western blot of recombinant GFP. Detection of His-tagged fusion GFP expressed in S. Choleraesuis. Membrane was incubated with a mixture of Anti-HisG-HRP antibody (1:3000) and Anti-His6-Peroxidase (1:3000) overnight. lane 1, total cell lysate of S. Choleraesuis expressing gfp under Ch promoter; lane 2, purified GFP expressed under TrcD promoter in S. Choleraesuis.
(Seleem et al., 2007a). The ‘A tract’ sequence (TrcD) had a positive effect on trc promoter activity in S. Choleraesuis, when positioned at −40 bp, increasing transcription 40% (Fig.1B) over the core trc promoter. There was no change in activity using A+T rich trcB promoter. This enhancement appears to be due to ability of the ‘A tract’ to provide binding site(s) for the RNA polymerase that contributes to the wrapping of the promoter DNA around the RNA polymerase (Aiyar et al., 1998; Seleem et al., 2007a). 3.3. RNA stem loop It has been shown that inserting a 6 bp RNA stem–loop at the appropriate place will prevent interaction or hybridization between the leader sequence and the downstream mRNA sequence leaving the leader sequence more accessible to ribosomal initiation, which leads to mRNA stabilization and enhancement of expression (Paulus et al., 2004). To further enhance the expression of heterologous genes in S. Choleraesuis, we inserted a tandem His-tag fusion that forms a RNA stem loop in the N-terminus of the mRNA (Seleem et al., 2007b). To test the effect of RNA stem loop on expression we applied the RNA stem– loop structure downstream of the strongest promoter (Ch) and the weakest promoter (groE). The RNA stem–loop enhanced expression almost 45% for Ch promoter (Fig. 1C) and 3 fold for groE promoter (Fig. 1D). We clearly demonstrate that a double His-tagged fusion with the predicted RNA stem–loop in the N-terminus of the mRNA enhanced the expression of proteins in S. Choleraesuis compared to the vectors that have a single His-tag fusion. 3.4. Protein purification and detection
Fig. 2. SDS-PAGE of purified recombinant GFP. SDS-PAGE of purified recombinant GFP from S. Choleraesuis using Ni-NTA Agarose (Qiagen) and 6 M Guanidine-HCL, stained with Coomassie blue, lane 1; total cell lysate of recombinant S. Choleraesuis; lane 2; elution, lane 3; purified recombinant GFP; M, Precision Plus Protein™ Dual Color Standards (Bio-Rad).
The His-tag fusion allowed one-step purification of the recombinant GFP from S. Choleraesuis by nickel chelate affinity chromatography. We were able to purify 380 μg of recombinant GFP using Ni-NTA Spin Columns and 8 M urea in comparison to 640 μg using Ni-NTA agarose and 6 M Guanidine-HCL. Using 6 M Guanidine-HCL was more efficient in solubilizing recombinant protein expressed in S. Choleraesuis. Although the Ni-NTA Spin Columns were very fast and easy to use, the protein purified with the Ni-NTA agarose was more pure (Fig. 2). The His-tag fusion protein was expressed and detected successfully in S. Choleraesuis. The Anti-His antibodies were used successfully to detect expression of GFP fused with the His-tag either in total cell lysates or purified protein (Fig. 3). In this study we tested a series of expression vectors for Salmonella that can be used for heterologous gene expression, protein detection and purification. The lacZ gene was cloned into the modified broad host range plasmid, transformed into S. Choleraesuis, expressed from various promoters. Recombinant GFP was detected by Western blot using Anti-His antibodies and purified using Ni-NTA. Expression and purification of recombinant proteins are fundamental techniques in molecular biology. Although, various expression systems are available and easy purification procedures of large amount of recombinant protein from E. coli are well established, they do not allow post-translational modification studies and protein–protein interaction in other microorganisms. Having an enhanced and simple system for expression and purification of recombinant protein directly from its native microorganism without using E. coli as an expression
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host will facilitate more accurate study of the characteristics of the expressed protein. Also, the system will overcome the problem that may arise from differences in codon usage, toxicity and signal sequence associated with the heterologous protein expressed in E. coli. We demonstrated one-step detection and purification of recombinant GFP directly from Salmonella spp. without using E. coli as an expression host. The creation of a synthetic RNA stem loop in the mRNA encoding the N-terminus of the target protein enhanced expression in Salmonella up to 3 fold, which can be attributed to the suppression of the long-range interaction between the translation initiation domain and gene-specific mRNA sequences and formation of accessible translational initiation domain (Paulus et al., 2004). The high level of protein expression achieved using these vectors did not alter the stability of the plasmid or the growth characteristics of the recombinant strains compared to the wild-type strains. Salmonella vaccine strains will benefit from the enhanced heterologous gene expression, as they can be used for the delivery of protective antigens of other pathogens (bivalent vaccines; (Cardenas and Clements, 1992). Although live vaccine strains containing an antibiotic-resistance-based plasmid selection systems are unacceptable for use in humans or animals, replacement by non-antibiotic resistance marker is possible (Curtiss et al., 1989; McNeill et al., 2000; Garmory et al., 2005). References Aiyar, S.E., Gourse, R.L., Ross, W., 1998. Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase alpha subunit. Proc. Natl. Acad. Sci. U. S. A. 95, 14652–14657. Amann, E., Brosius, J., Ptashne, M., 1983. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene 25, 167–178.
Antoine, R., Locht, C., 1992. Isolation and molecular characterization of a novel broadhost-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. Mol. Microbiol. 6, 1785–1799. Cardenas, L., Clements, J.D., 1992. Oral immunization using live attenuated Salmonella spp. as carriers of foreign antigens. Clin. Microbiol. Rev. 5, 328–342. Chen, C.Y., Winans, S.C., 1991. Controlled expression of the transcriptional activator gene virG in Agrobacterium tumefaciens by using the Escherichia coli lac promoter. J. Bacteriol. 173, 1139–1144. Curtiss III, R., Nakayama, K., Kelly, S.M., 1989. Recombinant avirulent Salmonella vaccine strains with stable maintenance and high level expression of cloned genes in vivo. Immunol. Invest. 18, 583–596. Estrem, S.T., Gaal, T., Ross, W., Gourse, R.L., 1998. Identification of an UP element consensus sequence for bacterial promoters. Proc. Natl. Acad. Sci. U. S. A. 95, 9761–9766. Garmory, H.S., Leckenby, M.W., Griffin, K.F., Elvin, S.J., Taylor, R.R., Hartley, M.G., Hanak, J.A., Williamson, E.D., Cranenburgh, R.M., 2005. Antibiotic-free plasmid stabilization by operator-repressor titration for vaccine delivery by using live Salmonella enterica Serovar Typhimurium. Infect. Immun. 73, 2005–2011. McNeill, H.V., Sinha, K.A., Hormaeche, C.E., Lee, J.J., Khan, C.M., 2000. Development of a nonantibiotic dominant marker for positively selecting expression plasmids in multivalent Salmonella vaccines. Appl. Environ. Microbiol. 66, 1216–1219. Paulus, M., Haslbeck, M., Watzele, M., 2004. RNA stem-loop enhanced expression of previously non-expressible genes. Nucleic Acids Res. 32, e78. Seleem, M.N., Vemulapalli, R., Boyle, S.M., Schurig, G.G., Sriranganathan, N., 2004. Improved expression vector for Brucella species. Biotechniques 37, 740–744. Seleem, M.N., Ali, M., Boyle, S.M., Mukhopadhyay, B., Witonsky, S.G., Schurig, G.G., Sriranganathan, N., 2006. Establishment of a gene expression system in Ochrobactrum anthropi. Appl. Environ. Microbiol. 72, 6833–6836. Seleem, M., Ali, M., Abd Al-Azeem, M.W., Boyle, S.M., Sriranganathan, N., 2007a. Highlevel heterologous gene expression in Ochrobactrum anthropi using an A-rich UP element. Appl. Microbiol. Biotechnol. 73, 1123–1127. Seleem, M., Ali, M., Al-Azeem, M.W., Boyle, S.M., Sriranganathan, N., 2007b. Enhanced expression, detection and purification of recombinant proteins using RNA stem loop and tandem fusion tags. Appl. Microbiol. Biotechnol. 75, 1385–1392. Vemulapalli, R., He, Y., Boyle, S.M., Sriranganathan, N., Schurig, G.G., 2000. Brucella abortus strain RB51 as a vector for heterologous protein expression and induction of specific Th1 type immune responses. Infect. Immun. 68, 3290–3296. Wang, Y., Mukhopadhyay, A., Howitz, V.R., Binns, A.N., Lynn, D.G., 2000. Construction of an efficient expression system for Agrobacterium tumefaciens based on the coliphage T5 promoter. Gene 242, 105–114.
Contents lists available at ScienceDirect
Gene j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g e n e
Methods paper
Vectors for enhanced gene expression and protein purification in Salmonella Mohamed N. Seleem a,c, Mohammed Ali b, Stephen M. Boyle c, Nammalwar Sriranganathan c,⁎ a
Institute for Critical Technology and Applied Science, Virginia Polytechnic Institute and State University, USA Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Assiut University, Egypt c Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, USA b
A R T I C L E
I N F O
Article history: Received 28 October 2007 Received in revised form 31 January 2008 Accepted 5 June 2008 Available online 22 June 2008
A B S T R A C T Improved expression vectors have been tested for protein expression studies in Salmonella spp. They are derived from the broad host range expression vector pNSGroE [Seleem, M.N., Vemulapalli, R., Boyle, S.M., Schurig, G.G. and Sriranganathan, N., 2004. Improved expression vector for Brucella species. Biotechniques 37, 740–744] and have several advantages (i) small in size (less than 3 kb); (ii) possess eleven unique restriction site to facilitate directional cloning; (iii) express proteins as His-tagged fusions for easy detection and purification; (iv) carry different promoters for various level of expression and (v) carry an UP element and RNA stem–loop for enhanced gene expression. We have demonstrated the ability of the new vectors to stably express heterologous proteins in Salmonella. We also demonstrated the utility of our vectors by detecting expression and purification of recombinant GFP. Our results suggest that these new vectors should improve gene expression in Salmonella, particularly those aimed at foreign antigen delivery. © 2008 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Several enhanced expression vectors have been constructed for cloning, gene expression, protein purification and promoter selection in O. anthropi (Seleem et al., 2006). The backbone of all vectors originated from the pNSGroE, a Brucella expression vector (Seleem et al., 2004) and a derivative of the pBBR1 originally isolated from Bordetella bronchiseptica (Antoine and Locht, 1992); the ability of the vectors to propagate in a wide variety of gram-negative bacteria encouraged us to test them in Salmonella. These vectors contain various promoters for different levels of expression and also His-tag fusion in the N-terminus to facilitate protein detection and purification. The functionality of all vectors was assessed by cloning the β-galactosidase gene and determining the level of protein expression and LacZ activity. We have exploited the transcriptional activity of the strong regulated coliphage T5 promoter (Wang et al., 2000), and trc hybrid promoter (Amann et al., 1983) in Salmonella. We were able to express, detect and purify recombinant proteins directly from Salmonella. We were able to further enhance gene expression in Salmonella by incorporation of three elements: an UP element, a RNA stem–loop and a tandem tag fusion (Estrem et al., 1998; Paulus et al., 2004). The expression vectors used in the current study proved to be easily transformed into and stably maintained in Salmonella.
2.1. Expression vectors
Abbreviations: CTD, Carboxy-terminal domain; GFP, Green fluorescence protein; LacZ, β-galactosidase gene; MCS, Multiple cloning site; UP, Upstream element. ⁎ Corresponding author. Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1410 Prices Fork Rd, Blacksburg, VA, 24061, USA. Tel.: +1 540 231 7171; fax: +1 540 231 3426. E-mail addresses: [email protected], [email protected] (N. Sriranganathan). 0378-1119/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2008.06.021
The sequences of all vectors, which are reported in this paper have been deposited in the GenBank database. Their description, sizes and accession numbers are listed in Table 1. 2.2. Expression of β-galactosidase (LacZ) In order to study the expression and activity of the cloned promoters inside Salmonella, the promoterless Escherichia coli βgalactosidase (lacZ) gene was amplified from pRSETB/β-gal as described before (Seleem et al., 2004) and cloned into all expression vectors downstream of the various promoters. 2.3. GFP constructs A promoterless Green Fluorescence Protein gene (gfp) was excised from pGFPuv vector (BD Biosciences Clontech) and cloned in-frame and down stream of the promoter in the multiple cloning site (MCS) of pNSTrcD and pNSCh. GFP was used as a visual marker for gene expression, Western blotting and protein purification. 2.4. Competent cells preparation and transformation To prepare Salmonella enterica serotype Choleraesuis (S. Choleraesuis) competent cells, 300 μl of culture (OD600 = 1) was spread on TSA plates, and incubated overnight at 37 °C. The bacteria were gently
96
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(Vemulapalli et al., 2000) were followed to determine and compare the levels of β-galactosidase expression from different promoters.
Table 1 Descriptions of the plasmids that were constructed and used in this study Plasmid
Size bp
Promoter
Type of expression
Accession number
2.5. Protein purification
pNSCh pNSKan pNSAmp pNST5 pNSTrcD pNSGroE pNSGroE2His pNSChE2His pNSTrcB pNSTrc
2903 2809 2891 2810 2937 2863 2881 2921 2938 2932
Ch r Kanr Ampr Phage T5 trc hybrid + UP B. abortus groE groE + RNA stem loop Ch + RNA stem loop trc hybrid + UP trc hybrid
Constitutive Constitutive Constitutive Regulated Regulated Constitutive Constitutive Constitutive Regulated Regulated
DQ412050 DQ412052 DQ412053 DQ412054 DQ412056 AY576605 DQ412049 DQ412051 DQ412057 DQ412055
The pNSTrcD-gfp expression vector with gfp expressed under the TrcD promoter was transformed into S. Choleraesuis as described earlier. The GFP was purified from recombinant S. Choleraesuis using 2 methods: a) Ni-NTA Spin Columns (Qiagen) under denaturing condition using 8 M urea and according to the manufacturer's instructions, 20% ethanol was added to the washing buffer to reduce contaminant proteins. b) Ni-NTA agarose (Qiagen) and 6 M guanidine HCl. 20 mM imidazol and 20 mM β-mercaptoethanol were added to the lysis buffer and 1% Triton X-100 was added to the washing buffer to reduce contaminating proteins. The concentration of the purified recombinant protein was determined by BCA Protein Assay kit (Pierce) using the manufacturer's enhanced test tube procedure after removal of urea by Microcon centrifugal filter devices YM-10 (Millipore). The purity of the extracted protein was estimated following visualization on SDS-PAGE.
scraped from the plates and washed twice with ice-cold 10% glycerol. The cells were resuspended to a density of approximately 1011 cells/ml in icecold 10% glycerol. The plasmids containing either lacZ or gfp were transformed into S. Choleraesuis by electrotransformation with a Gene Pulser (BTX) set at 2.7 kV, 25 μF and 200 Ω using a 1 mm gap cuvette (Eppendorf). After electroporation, the cells were transferred to SOC media (Invitrogen) and incubated at 37 °C, with shaking at 200 rpm, for 2 h to allow expression of the antibiotic resistance gene. The transformation mixture was plated on several TSA plates containing 30 μg chloramphenicol. Transformation efficiency and stability of the plasmids were calculated as described before (Seleem et al., 2004). Previously described procedures
2.6. Western blot To test the fusion of the His-epitope tagging and efficiency of detection, Western blot analysis was performed by separating the total cell lysates of recombinant S. Choleraesuis expressing GFP and 15 μg of purified recombinant GFP on a 12% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was incubated overnight
Fig. 1. LacZ activity from different promoters in S. Choleraesuis. Promoter activity of each construct was determined and represented as Miller unit. The data are means ± SD of triplicate assays. A, activity of lacZ under different promoter in S. Choleraesuis; B, effect of UP element on the expression of trc promoter; C and D, effect of RNA stem–loop on Ch and groE promoter.
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with a mixture of Anti-HisG-HRP antibody (1:3000) (Invitrogen) and Anti-His6-Peroxidase (Roche) (1:3000). The combination of antibodies that recognize 6×His and one glycine (Anti-HisG-HRP) with antibodies that recognize only 6×His (Anti-His6-Peroxidase) produced more intense band and higher sensitivity of detection. 3. Results and discussion All expression vectors were successfully introduced into S. Choleraesuis with an efficiency of 4.5 × 104 transformants per μg DNA. All plasmids were found to be stably maintained after 8 serial passages over 16 days on TSA plates in the absence of chloramphenicol selection (Seleem et al., 2004). All plasmids constructs were successfully transformed and stably maintained in S. Typhimurium, S. Dublin and S. Newport. 3.1. Promoter activity A comparison was made between the relative levels of LacZ activity expressed under different promoters. All tested promoters were able to express inside S. Choleraesuis with various strengths (Fig. 1A). The chloramphenicol promoter (Ch promoter) was the strongest promoter. In this study, we have exploited the transcriptional activity of the strong coliphage T5 promoter (Wang et al., 2000) and trc hybrid promoter (Amann et al., 1983) in S. Choleraesuis and demonstrated that both promoters are functional (Fig. 1A). The T5 promoter and trc hybrid promoter have the lac operator (lacO), that permits binding of the lac repressor to regulate transcription in bacterial strains that harbor the lacIq gene. Regulated expression using the T5 promoter and trc hybrid promoter in S. Choleraesuis is possible by incorporating the lacIq gene into the plasmid (Chen and Winans, 1991). 3.2. UP element DNA regions that flank a gene's promoter play an important role in determining transcription efficiency by interacting with the carboxyterminal domain (CTD) of RNA polymerase α-subunit (Estrem et al.,1998). We placed an adenine (A)-rich and A+T rich upstream element (UP) (Estrem et al., 1998) between −40 and −59 bp of the core trc promoter
Fig. 3. Western blot of recombinant GFP. Detection of His-tagged fusion GFP expressed in S. Choleraesuis. Membrane was incubated with a mixture of Anti-HisG-HRP antibody (1:3000) and Anti-His6-Peroxidase (1:3000) overnight. lane 1, total cell lysate of S. Choleraesuis expressing gfp under Ch promoter; lane 2, purified GFP expressed under TrcD promoter in S. Choleraesuis.
(Seleem et al., 2007a). The ‘A tract’ sequence (TrcD) had a positive effect on trc promoter activity in S. Choleraesuis, when positioned at −40 bp, increasing transcription 40% (Fig.1B) over the core trc promoter. There was no change in activity using A+T rich trcB promoter. This enhancement appears to be due to ability of the ‘A tract’ to provide binding site(s) for the RNA polymerase that contributes to the wrapping of the promoter DNA around the RNA polymerase (Aiyar et al., 1998; Seleem et al., 2007a). 3.3. RNA stem loop It has been shown that inserting a 6 bp RNA stem–loop at the appropriate place will prevent interaction or hybridization between the leader sequence and the downstream mRNA sequence leaving the leader sequence more accessible to ribosomal initiation, which leads to mRNA stabilization and enhancement of expression (Paulus et al., 2004). To further enhance the expression of heterologous genes in S. Choleraesuis, we inserted a tandem His-tag fusion that forms a RNA stem loop in the N-terminus of the mRNA (Seleem et al., 2007b). To test the effect of RNA stem loop on expression we applied the RNA stem– loop structure downstream of the strongest promoter (Ch) and the weakest promoter (groE). The RNA stem–loop enhanced expression almost 45% for Ch promoter (Fig. 1C) and 3 fold for groE promoter (Fig. 1D). We clearly demonstrate that a double His-tagged fusion with the predicted RNA stem–loop in the N-terminus of the mRNA enhanced the expression of proteins in S. Choleraesuis compared to the vectors that have a single His-tag fusion. 3.4. Protein purification and detection
Fig. 2. SDS-PAGE of purified recombinant GFP. SDS-PAGE of purified recombinant GFP from S. Choleraesuis using Ni-NTA Agarose (Qiagen) and 6 M Guanidine-HCL, stained with Coomassie blue, lane 1; total cell lysate of recombinant S. Choleraesuis; lane 2; elution, lane 3; purified recombinant GFP; M, Precision Plus Protein™ Dual Color Standards (Bio-Rad).
The His-tag fusion allowed one-step purification of the recombinant GFP from S. Choleraesuis by nickel chelate affinity chromatography. We were able to purify 380 μg of recombinant GFP using Ni-NTA Spin Columns and 8 M urea in comparison to 640 μg using Ni-NTA agarose and 6 M Guanidine-HCL. Using 6 M Guanidine-HCL was more efficient in solubilizing recombinant protein expressed in S. Choleraesuis. Although the Ni-NTA Spin Columns were very fast and easy to use, the protein purified with the Ni-NTA agarose was more pure (Fig. 2). The His-tag fusion protein was expressed and detected successfully in S. Choleraesuis. The Anti-His antibodies were used successfully to detect expression of GFP fused with the His-tag either in total cell lysates or purified protein (Fig. 3). In this study we tested a series of expression vectors for Salmonella that can be used for heterologous gene expression, protein detection and purification. The lacZ gene was cloned into the modified broad host range plasmid, transformed into S. Choleraesuis, expressed from various promoters. Recombinant GFP was detected by Western blot using Anti-His antibodies and purified using Ni-NTA. Expression and purification of recombinant proteins are fundamental techniques in molecular biology. Although, various expression systems are available and easy purification procedures of large amount of recombinant protein from E. coli are well established, they do not allow post-translational modification studies and protein–protein interaction in other microorganisms. Having an enhanced and simple system for expression and purification of recombinant protein directly from its native microorganism without using E. coli as an expression
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host will facilitate more accurate study of the characteristics of the expressed protein. Also, the system will overcome the problem that may arise from differences in codon usage, toxicity and signal sequence associated with the heterologous protein expressed in E. coli. We demonstrated one-step detection and purification of recombinant GFP directly from Salmonella spp. without using E. coli as an expression host. The creation of a synthetic RNA stem loop in the mRNA encoding the N-terminus of the target protein enhanced expression in Salmonella up to 3 fold, which can be attributed to the suppression of the long-range interaction between the translation initiation domain and gene-specific mRNA sequences and formation of accessible translational initiation domain (Paulus et al., 2004). The high level of protein expression achieved using these vectors did not alter the stability of the plasmid or the growth characteristics of the recombinant strains compared to the wild-type strains. Salmonella vaccine strains will benefit from the enhanced heterologous gene expression, as they can be used for the delivery of protective antigens of other pathogens (bivalent vaccines; (Cardenas and Clements, 1992). Although live vaccine strains containing an antibiotic-resistance-based plasmid selection systems are unacceptable for use in humans or animals, replacement by non-antibiotic resistance marker is possible (Curtiss et al., 1989; McNeill et al., 2000; Garmory et al., 2005). References Aiyar, S.E., Gourse, R.L., Ross, W., 1998. Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase alpha subunit. Proc. Natl. Acad. Sci. U. S. A. 95, 14652–14657. Amann, E., Brosius, J., Ptashne, M., 1983. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene 25, 167–178.
Antoine, R., Locht, C., 1992. Isolation and molecular characterization of a novel broadhost-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. Mol. Microbiol. 6, 1785–1799. Cardenas, L., Clements, J.D., 1992. Oral immunization using live attenuated Salmonella spp. as carriers of foreign antigens. Clin. Microbiol. Rev. 5, 328–342. Chen, C.Y., Winans, S.C., 1991. Controlled expression of the transcriptional activator gene virG in Agrobacterium tumefaciens by using the Escherichia coli lac promoter. J. Bacteriol. 173, 1139–1144. Curtiss III, R., Nakayama, K., Kelly, S.M., 1989. Recombinant avirulent Salmonella vaccine strains with stable maintenance and high level expression of cloned genes in vivo. Immunol. Invest. 18, 583–596. Estrem, S.T., Gaal, T., Ross, W., Gourse, R.L., 1998. Identification of an UP element consensus sequence for bacterial promoters. Proc. Natl. Acad. Sci. U. S. A. 95, 9761–9766. Garmory, H.S., Leckenby, M.W., Griffin, K.F., Elvin, S.J., Taylor, R.R., Hartley, M.G., Hanak, J.A., Williamson, E.D., Cranenburgh, R.M., 2005. Antibiotic-free plasmid stabilization by operator-repressor titration for vaccine delivery by using live Salmonella enterica Serovar Typhimurium. Infect. Immun. 73, 2005–2011. McNeill, H.V., Sinha, K.A., Hormaeche, C.E., Lee, J.J., Khan, C.M., 2000. Development of a nonantibiotic dominant marker for positively selecting expression plasmids in multivalent Salmonella vaccines. Appl. Environ. Microbiol. 66, 1216–1219. Paulus, M., Haslbeck, M., Watzele, M., 2004. RNA stem-loop enhanced expression of previously non-expressible genes. Nucleic Acids Res. 32, e78. Seleem, M.N., Vemulapalli, R., Boyle, S.M., Schurig, G.G., Sriranganathan, N., 2004. Improved expression vector for Brucella species. Biotechniques 37, 740–744. Seleem, M.N., Ali, M., Boyle, S.M., Mukhopadhyay, B., Witonsky, S.G., Schurig, G.G., Sriranganathan, N., 2006. Establishment of a gene expression system in Ochrobactrum anthropi. Appl. Environ. Microbiol. 72, 6833–6836. Seleem, M., Ali, M., Abd Al-Azeem, M.W., Boyle, S.M., Sriranganathan, N., 2007a. Highlevel heterologous gene expression in Ochrobactrum anthropi using an A-rich UP element. Appl. Microbiol. Biotechnol. 73, 1123–1127. Seleem, M., Ali, M., Al-Azeem, M.W., Boyle, S.M., Sriranganathan, N., 2007b. Enhanced expression, detection and purification of recombinant proteins using RNA stem loop and tandem fusion tags. Appl. Microbiol. Biotechnol. 75, 1385–1392. Vemulapalli, R., He, Y., Boyle, S.M., Sriranganathan, N., Schurig, G.G., 2000. Brucella abortus strain RB51 as a vector for heterologous protein expression and induction of specific Th1 type immune responses. Infect. Immun. 68, 3290–3296. Wang, Y., Mukhopadhyay, A., Howitz, V.R., Binns, A.N., Lynn, D.G., 2000. Construction of an efficient expression system for Agrobacterium tumefaciens based on the coliphage T5 promoter. Gene 242, 105–114.