Journal of Virological Methods 196 (2014) 56–64
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
High-yield soluble expression of recombinant influenza virus antigens from Escherichia coli and their potential uses in diagnosis Yo Han Jang a , Seung Hee Cho a , Ahyun Son a , Yun Ha Lee a , Jinhee Lee a , Kwang-Hee Lee a , Baik Lin Seong a,b,∗ a b
Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, South Korea Translational Research Center for Protein Function Control, Yonsei University, Seoul, South Korea
a b s t r a c t Article history: Received 17 April 2013 Received in revised form 16 October 2013 Accepted 22 October 2013 Available online 8 November 2013 Keywords: Influenza viruses Recombinant proteins Bacteria Antibody library Fusion partner ELISA
Although antiviral drugs and vaccines have been successful for mitigating influenza virus infections, the lack of general technical platform for the timely supply of soluble and highly purified influenza viral antigens presents a serious bottleneck for the subsequent analysis for the effective control of the viral disease. Using the Escherichia coli (E. coli) lysyl tRNA synthetase (LysRS) as a novel fusion partner, this study reports the soluble expression of influenza viral proteins in E. coli host, construction of antibody library against the virus, and detection of anti-influenza antibodies using the expressed viral antigens. When influenza A and B viral proteins were fused with the LysRS, the fusion proteins were expressed predominantly as soluble forms and their production yields were high enough to be amenable to immunization protocols in rabbits for antibody generation. The produced antibodies showed high level binding specificity against the respective viral proteins, with cross-reactivity across heterologous viruses within the same type of influenza viruses. In addition, LysRS-HA fusion protein could bind specifically to anti-HA antibodies in the virus-infected mouse serum in widely accepted two detection methods, Western blot and ELISA. These results present a convenient tool for the production of antibodies specific to the virus as well as the rapid detection of influenza viral infections, ultimately contributing to the control of influenza viruses including highly pathogenic H5N1, pandemic H1N1, or the recent H7N9 influenza viruses. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Influenza virus poses persistently serious threats to human health, which become more pronounced in the event of sporadic pandemics by human-to-human transmission of highly pathogenic viruses (Potter, 2001; Nicholson et al., 2003). While inactivated killed vaccines and cold-adapted live attenuated vaccines can be prepared from cultured influenza viruses, the development of recombinant protein vaccines or in vitro antiviral drug or target screenings need a substantial amount of soluble and highly purified viral antigens. Although Escherichia coli systems remain the most preferred choice for the over-expression of heterologous proteins due to distinct advantages including inexpensive carbon source requirements, rapid growth rates, and the simplicity of scale-up (Baneyx, 1999), influenza viral proteins have been hardly amenable to soluble expression in this host, and, in particular, it has been
∗ Corresponding author at: Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, South Korea. Tel.: +82 2 2123 2885; fax: +82 2 362 7265. E-mail addresses: [email protected], [email protected] (B.L. Seong). 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.10.035
considered extremely difficult to express transmembrane proteins, such as hemagglutinin (HA), neuraminidase (NA), and M2 ion channel protein, as soluble forms. With maltose-binding protein (MBP), N-utilizing substance-A (NusA), and glutathione S-transferase (GST) at the head of list (Smith and Johnson, 1988; Davis et al., 1999; Fox and Waugh, 2003; Nallamsetty and Waugh, 2006), a number of fusion partners have been advanced as solubility enhancers for the bacterial expression of aggregation-prone proteins (Young et al., 2012). However, most of the fusion partners still failed to support soluble expression for target proteins (Esposito and Chatterjee, 2006), underlining the need for a novel fusion partner that has general versatility in broad-spectrum. Recently, it was reported that RNA-binding proteins (RBP) could serve as novel fusion partners that significantly accelerated the folding and solubility of passenger proteins in E. coli (Kim et al., 2007; Choi et al., 2008). Consistent with several reports that RNA had accelerated the folding of interacting proteins (Frankel and Smith, 1998; Rentzeperis et al., 1999; Uversky et al., 2000), interaction with RNA stimulated the folding of the RBPs themselves and also promoted the folding and soluble expression of their fused aggregation-prone proteins (Choi et al., 2008). In addition, the RBP demonstrated superior efficiency in gaining
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and enhancing the solubility to MBP, a widely used fusion partner (Choi et al., 2008), exhibiting a potential to serve as an alternative powerful tool for recombinant protein expression. With a view to generate antibody library against the influenza viruses, the RBP fusion strategy was applied into the soluble expression and purification of influenza internal and surface membrane proteins by fusion to E. coli lysyl tRNA synthetase (LysRS). The prominent ability of the LysRS fusion to induce high yield soluble expression of influenza viral proteins presents significant benefits for the production of viral antigen or antibody libraries for analytical, therapeutic, prophylactic, and diagnostic purposes in numerous non-clinical and clinical investigations. 2. Materials and methods 2.1. Construction of protein expression vectors pGE-LysRS4 vector constructed in previous work (Choi et al., 2008) was used as a vector for the expression of influenza viral proteins in E. coli. The LysRS expression cassette includes LysRS, D6 hexa-aspartic acids linker, TEV recognition site, multicloning site, and the hexa-histidines tag under the T7 promoter (Fig. 1A). The cDNAs each encoding the complete open reading frame (ORF) of individual viral proteins from A/WSN/33 (H1N1), A/Puerto Rico/8/34 (H1N1), and B/Yamagata/16/88 were amplified by reverse transcription PCR (RT-PCR) and then inserted into the pGE-LysRS4 vector. 2.2. Protein expression Protein expression and SDS-PAGE analysis were performed as described previously (Choi et al., 2008). Each expression plasmid was transformed into the E. coli expression host, BL21 (DE3) pLysS. A single colony of transformants was inoculated into 3 ml of LB containing both 50 g/ml ampicillin and 34 g/ml chloramphenicol. The culture medium was then diluted into 25 ml of the fresh LB containing the same antibiotics and the cells were cultured to the optical density (OD) of 0.5 at 600 nm. Expression of proteins was induced for 6 h at 20–30 ◦ C by the addition of 1 mM IPTG. After centrifugation, the cell pellets were suspended in 0.3 ml of PBS and disrupted by sonication. The cell lysates were centrifuged at 11,000 × g for 12 min and separated into soluble and pellet fractions. Each fraction was then mixed with the same volume of 2× SDS loading buffer and boiled. These samples were subjected to SDS-PAGE and visualized by staining with Coomassie brilliant blue R-250.
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bovine serum albumin (BSA) (Amresco, Solon, OH, USA) of a known concentration as control. 2.4. Animal cells and influenza viruses Madin–Darby canine kidney (MDCK) cells were maintained in minimal essential medium supplemented with 10% fetal bovine serum. To evaluate the binding specificities of antisera collected from the rabbits immunized with the fusion proteins, MDCK cells were infected with 1 multiplicity of infection (MOI) of each of four influenza A viruses, A/WSN/33 (H1N1), A/Singapore/6/86 (H1N1), A/Sydney/5/97 (H3N2), A/Puerto Rico/8/34 (H1N1), and of three influenza B viruses; B/Lee/40, B/Yamagata/16/88, and cold-adapted B/Lee/40 (Seo et al., 2008), and the cells were harvested 24 h after the infections for Western blots. 2.5. Immunization and polyclonal antibodies production in rabbits Rabbit experiments were conducted in the animal facilities of the LabFrontier (Anyang, South Korea). To obtain polyclonal antibodies specific to influenza viral proteins, two rabbits were immunized with each of purified LysRS-fused recombinant influenza viral proteins. Each 0.5 mg of LysRS-fused protein emulsified with Freund’s complete adjuvant was injected intradermally at multiple sites on the back of each rabbit. Starting from four weeks after the first immunization, each animal was boosted twice by an additional 0.2 mg of the protein and adjuvant mixtures with the interval of two weeks. Sera samples were collected at seven days after each boosting and the final sera samples were aliquoted and stored at −80 ◦ C. The experimental protocol was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) of LabFrontier. 2.6. Mice infection and serum preparation 6-Week-old female BALB/c mice were infected intranasaly with 106 PFU of attenuated mutant strain of A/Puerto Rico/8/34 (H1N1) virus (Jang et al., 2013) or mock-infected with PBS as a control. Three weeks later the mice blood samples were collected from the mice via retro-orbital bleeding and clotted at 4 ◦ C overnight to collect sera samples. Mouse study was carried out in strict accordance with the recommendations of the Korean Food and Drug Administration (KFDA) guidelines. Protocols were reviewed and approved by the IACUC of the Yonsei Laboratory Animal Research Center (YLARC) (Permit number: 2012-0094). 2.7. ELISA
2.3. Purification and quantification of fusion proteins Proteins were purified from 1 l culture of each transformant using nickel affinity chromatography. The harvested cell pellets were suspended in 5 ml of equilibration buffer A (20 mM Tris–HCl (pH 7.5), 300 mM NaCl, 10% glycerol, 2 mM 2-mercaptoethanol, and 5 mM imidazole) and disrupted by sonication. The soluble fractions were recovered by centrifugation at 11,000 × g for 20 min and then applied onto HiTrap chelating HP column (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK). After washing, proteins were eluted with 50 ml of elution buffer with the same composition of equilibration buffer as above except for the linear gradients of imidazole ranging from 10 mM to 300 mM. The fractions containing proteins of interest were pooled, concentrated, dialyzed against the buffer containing 50 mM Tris–HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, and 1 mM DTT in 50% glycerol. After SDS-PAGE, the purified proteins were quantified by gel densitometry scanning of the Coomassie-blue stained protein band using
Microtiter 96-well plates were coated with 100 ng/well of LysRS-fused influenza viral protein and incubated at 4 ◦ C overnight. The plates were washed and blocked by 1% BSA for 1 h at room temperature (RT). 100 l of sera with various dilutions were added to the wells for 1 h, and the same volume of a secondary goat anti-rabbit IgG antibody or goat-anti mouse IgG antibody conjugated with HRP (Sigma–Aldrich, St. Louis, MO, USA) was treated for 1 h. After washings, substrate solution was added to the well and the plate was incubated in the dark. The colorimetric reaction was stopped by the addition of 100 l/well of 1 M H2 SO4 and the absorbance was read at 492 nm on ELISA reader (FLUOstar OPTIMA, BMG LABTECH, Offenburg, Germany). 2.8. Western blot analysis MDCK cells infected with one MOI of each influenza virus were subjected to Western blot analysis using each final serum
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Fig. 1. Soluble expression of LysRS-fused influenza A or B viral proteins in E. coli. (A) Schematic illustration of pGE-LysRS4 vector used for the expression of fusion proteins in E. coli. (B) Expression profiles of ten fusion proteins in soluble or insoluble fraction. The fusion proteins were expressed at 27 ◦ C (for all proteins except for A-NA) or 20 ◦ C (for A-NA) and their solubility was analyzed by SDS-PAGE. M, T, S, and P represent molecular weight marker in kDa, total lysates, soluble fraction, and insoluble fraction, respectively. Arrows indicate the induced fusion proteins. (C) Solubility of each of fusion proteins in B estimated by gel densitometer.
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produced from immunized rabbits. Various dilutions of each serum were tested in the analysis to determine optimal dilution, which was presented in results. As secondary antibodies, we used goat anti-rabbit IgG monoclonal antibody conjugated with horseradish peroxidase (Sigma–Aldrich, St. Louis, MO, USA). For the detection of anti-HA and anti-NA antibodies in influenza virus-infected mouse sera, 500 ng of LysRS-HA and LysRS-NA fusion proteins were loaded onto SDS-PAGE, which were then subjected to Western blot with the mouse serum with a dilution of 1:500. 3. Results 3.1. Soluble expression of LysRS-fused influenza viral proteins Full-length five viral proteins (including three internal proteins, M1, NS1, and NS2, and two surface membrane proteins NA and M2) of A/WSN/33 (H1N1) virus and HA protein of A/Puerto Rico/8/34 (H1N1) were subcloned into pGE-LysRS4 vector as a fusion to E. coli lysyl tRNA synthetase (LysRS). Similarly, the four proteins (M1, M2, NS1, and NS2) of B/Yamagata/16/88 virus were subcloned into the same expression vector. All fusion proteins were tested for soluble expression in E. coli host, BL21 (DE3) pLysS (Fig. 1A). Protein expression was induced by the addition of 1 mM IPTG under various temperature conditions, and the cell lysates were fractionated into soluble supernatants and insoluble pellets to determine the solubility of the expressed proteins. Through repetitive screenings of temperature and solubility, it was found that the most of the fusion proteins were expressed predominantly as soluble forms at 27 ◦ C. Interestingly, the expression level of full-length HA protein was poor, but truncation of 35 amino acids residue encoding C-terminal transmembrane and cytoplasmic domain dramatically increased the expression level of the HATM protein (Fig. 1B). Overall, the solubility was ≥80% for all proteins expressed (Fig. 1C). The high solubility of fusion proteins at relatively lower temperatures was consistent with previous results that the solubility of RBP-fused proteins increased substantially at lower temperature (Choi et al., 2008). Of note, the surface membrane protein NA of A/WSN/33 (H1N1) virus was most soluble at 20 ◦ C (Fig. 1B). Likewise, transmembrane proteins, A-M2 and B-M2, were also successfully expressed as soluble forms (Fig. 1B). Thus, the LysRS demonstrated prominent ability to induce the soluble expression of its linked protein, regardless of whether the cargo is an internal or transmembrane protein. 3.2. Scale-up and purification of fusion proteins Expressed fusion proteins were purified from 1 l culture of each transformant using one-step nickel chromatography. After cell lysis and fractionation, soluble fractions that contained the most of expressed fusion proteins were applied onto a column and eluted with linear gradients of imidazole ranging 10–300 mM. Hexa-histidines-tagged fusion proteins were purified efficiently from crude extract with high level homogeneity, albeit with the minor mixtures of probably E. coli-derived histidine-rich proteins or degraded fragments from the fusion proteins (Fig. 2A). The SDS-PAGE analysis of protein profiles at different stages of purification step showed that the most of each purified fusion protein migrated with expected molecular mass, indicating that, despite their relatively large molecular size ranging from 72 kDa to 114 kDa, the fusion proteins remained highly soluble during the scale-up expression and subsequent purification steps. The elution fractions containing the target fusion protein were pooled and dialyzed. Each of purified fusion proteins was subjected to SDS-PAGE analysis and quantified by comparing the band density with BSA of known concentration by gel densitometer scanning (Fig. 2B). The final yields of
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the fusion proteins were in the range of 1–2 mg/ml. Taken together, the LysRS showed excellent solubilizing and stabilizing ability during the expression and purification of influenza viral proteins in E. coli. 3.3. Antibody production against influenza viral proteins Among the ten fusion proteins expressed, five fusion proteins derived from influenza A/WSN/33 (H1N1) virus were used for antibody production from rabbits. Rabbits were primarily immunized with each fusion protein, and four weeks later the rabbits were boosted two times in two weeks interval. To estimate the level of specific antibody contained in the final serum, ELISA was performed using the fusion proteins as coated antigens. As compared to pre-immune sera, all five sera samples contained high level antibodies, with strong ELISA response even after 100,000fold dilution (Fig. 3A). The ELISA results may represent antibody responses against either LysRS or viral protein, or both, and thus do not reflect the exact levels of viral protein-specific antibodies. Therefore, Western blot analysis was performed on influenzainfected cells to examine whether the produced antibodies could bind specifically to their respective target viral proteins. MDCK cells were infected with 1 MOI of various influenza A and B virus strains, and the cell lysates were subjected to Western blot analysis to investigate the breadth of specificities of the antibodies. All of the five antibodies bound specifically to the viral proteins of influenza A viruses but not to those of B viruses (Fig. 3B). Although the fusion proteins were derived from H1N1 strain, the antibodies showed cross-reactivity against heterologous H1N1 and H3N2 strains. The cross-reactivity observed in the M1, M2, NS1, and NS2 was reasonably predictable since those proteins were highly conserved within the same type viruses. However, the cross reactivity shown in the NA antibody was remarkable, considering that the sequence homology of the NA proteins between A/WSN/33 (H1N1) and A/Sydney/5/97 (H3N2) was substantially low (41% in identities and 57% in positives). Thus, the antibodies exhibited a wide range of binding specificities within the influenza A type viruses against conserved internal proteins as well as highly variable surface antigen. 3.4. Detection of anti-influenza antibodies in infected mouse using soluble viral proteins Besides the production of antibodies specific to the viral antigens, highly soluble and purified viral proteins can be used for the detection of specific antibodies in virus-infected animals and humans. To explore this potential, the mice were infected with 106 PFU of the attenuated strain of A/Puerto Rico/8/34 (H1N1) (Jang et al., 2013) and the serum samples were tested for the presence of virus-specific antibodies using the two most popular detection tools, Western blot and ELISA. In the Western blot, anti-HA antibodies in all the infected mice sera were detected easily by the LysRS-HATM fusion protein. However, the anti-NA antibodies were barely detectable by the LysRS-NA proteins, with only one of four mice serum generating observable band (Fig. 4A). Likewise, when 100 ng/well of each fusion protein was coated into a microtiter plate, ELISA produced high level detection signal to LysRS-HATM protein in all the vaccinated mice sera applied, whereas the signal to LysRS-NA protein was nearly negative with similar absorbance to the LysRS control and PBS group (Fig. 4B). Increase in the concentration of the LysRS-NA protein did not produce any noticeable difference as compared to the controls in both assays. Considering the efficient production of anti-NA antibodies in the rabbits immunized with LysRS-NA protein (Fig. 3), poor detection ability of the LysRS-NA proteins can be reasonably attributed to much lower abundance of anti-NA antibodies in the
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Fig. 2. Purification of influenza A or B viral fusion proteins. (A) SDS-PAGE analysis showing protein profiles during the purification step. M, protein size marker; L, lysate of soluble fraction; F, unbound flow through; W, washing step. Arrows indicate the target fusion proteins. (B) SDS-PAGE analysis of each of pooled fusion proteins from A. M, protein size marker (kDa). Arrows indicate the induced fusion proteins.
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Fig. 3. Antibody productions against the influenza A viral proteins. (A) The antibodies levels produced in the rabbits immunized with LysRS-fused influenza A viral proteins. Serum IgG antibodies levels in pre-immune or final serum were estimated by ELISA using fusion viral proteins as coated antigens. The result of one of the two immunized rabbits was shown. (B) Binding specificities of antibodies against influenza viral proteins. MDCK cells were infected with 1 MOI of each of influenza A or B viruses, or mock-infected, and the cells were harvested at 24 h after the infections. The cell lysates were subjected to Western blot analysis using each final antiserum with an indicated dilution.
vaccinated mice sera relative to anti-HA antibodies (Kilbourne et al., 1987; Feng et al., 2009). Taken together, these results show that the E. coli-expressed influenza HATM protein can serve as useful detection tools for measuring the quantity or quality of antibody responses to the viruses.
4. Discussion In this study antigen and antibody libraries were established against influenza viruses using E. coli expression system, in which the LysRS was employed as a novel fusion partner to successfully
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Fig. 4. Detection of anti-influenza antibodies in virus-infected mice sera using LysRS-fused viral proteins. (A) Western blot analysis for the detection of anti-influenza surface antigen antibodies. Mice were infected with 106 PFU of mutant strain of A/Puerto Rico/8/34 (H1N1) (n = 4) or mock-infected with PBS (n = 1), and three weeks later sera were collected from the mice. 500 ng of LysRS, LysRS-HATM, and LysRS-NA proteins were run by SDS-PAGE and then subjected to Western blot with the each mouse serum of a dilution of 1:500. () Indicates the band of full-length fusion protein, LysRS-HATM (114 kDa) or LysRS-NA (111 kDa). (B) ELISA for the detection of anti-influenza surface antigen antibodies. Each microtiter 96-well plate was coated with 100 ng/well of LysRS, LysRS-HATM, or LysRS-NA protein, and the twofold serial dilutions of the serum of vaccinated (V, n = 5) or nonvaccinated (P, n = 3) mice were applied onto the plate for the analysis. Scanned images of the plates after the analysis are shown (upper), and their measured optical density (OD) of each mouse serum for each coated antigen protein are shown in the same graph (lower).
express all viral proteins as soluble forms. The specificity of antibodies is consistent with the serological classification of influenza viruses (Krug, 2001), which attests to the quality of antigens produced in this report. Previously, the full-length influenza viral
proteins have been refractory to soluble expression in E. coli host, with potential exception for the NP proteins (Huang et al., 2012). More notably, the bacterial expressions of the transmembrane proteins of influenza virus, such as HA, NA, and M2, showed low
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efficiency even when expressed and recovered from insoluble pellets (Davis et al., 1981; Jones and Brownlee, 1985; Ebrahimi et al., 2010), consequently leading to the changes of expression system to relatively expensive eukaryotic hosts, especially for recombinant vaccines based on the HA or M2 (Treanor et al., 1996; Wei et al., 2008; Bosch et al., 2010; Murugan et al., 2013). As the first report of the soluble expression of full-length influenza viral proteins in E. coli, these results present a generally applicable toolbox for the establishment of antigen and antibody libraries of human viruses and their uses in the detection assays as well. Previous study showed that most of aggregation-prone human proteins tested were expressed as soluble forms when fused with the LysRS, which were much more efficient than MBP fusion (Choi et al., 2008). Robust folding and solubility enhancement is now extended to human-infecting viral proteins as exemplified by the influenza proteins in the present study. Notably, the solubility enhancement was robust also for the transmembrane proteins, e.g., HA, NA, or M2, which usually aggregate via hydrophobic interactions in the membrane spanning region. These data together demonstrate that the present fusion system may offer a high-throughput soluble expression platform of proteins of eukaryotic or viral origin, overcoming technical difficulties still persisting in fusion technologies (Esposito and Chatterjee, 2006). Substantial amount of soluble and purified viral proteins is prerequisite for the generation of specific antibodies as well as highly sensitive detection methods, for which the present strategy met the demand successfully. In addition, the production of soluble viral antigen may provide better opportunity to control the viral diseases by the development of recombinant vaccines or antiviral drug screening in vitro. E. coli expression systems have been used for the production of recombinant influenza vaccines, but limited only to sub-domain of the HA or M2, due to low expression and inclusion body formation of the whole proteins (Neirynck et al., 1999; Ernst et al., 2006; Aguilar-Yanez et al., 2010; Khurana et al., 2010; DuBois et al., 2011; Khurana et al., 2011). On the other hand, the soluble expression of full-length NA and M2 proteins by fusion with the LysRS may be very helpful for developing recombinant vaccine spanning all antigenic epitopes required for sufficient protective efficacy. Influenza viruses exhibit extremely diverse antigenicity comprising various combinations of 16 HA and 9 NA subtypes (Fouchier et al., 2005). The importance of a timely supply of soluble antigens and corresponding antibodies cannot be overstated in the epidemiological studies of influenza viruses, especially considering the zoonotic nature of influenza transmission. The present results therefore could be extended to other influenza strains, including H5N1 highly pathogenic avian influenza (Vijaykrishna et al., 2008), 2009 pandemic H1N1 influenza (Neumann et al., 2009), and the recent 2013 H7N9 strains with high mortality or morbidity among human population (Uyeki and Cox, 2013). While further extending the utility of RNAinteraction mediated protein folding for recombinant proteins (Choi et al., 2009, 2011, 2012), the present results are expected to provide practical means to establish antigen and antibody libraries for infectious agents for analytical, diagnostic, and prophylactic purposes.
Role of the funding source This work was supported by a grant from the Korea Healthcare Technology R&D project of Ministry for Health, Welfare and Family Affairs of Republic of Korea [Grant number A085105]. The funding source did not have roles in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
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Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
High-yield soluble expression of recombinant influenza virus antigens from Escherichia coli and their potential uses in diagnosis Yo Han Jang a , Seung Hee Cho a , Ahyun Son a , Yun Ha Lee a , Jinhee Lee a , Kwang-Hee Lee a , Baik Lin Seong a,b,∗ a b
Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, South Korea Translational Research Center for Protein Function Control, Yonsei University, Seoul, South Korea
a b s t r a c t Article history: Received 17 April 2013 Received in revised form 16 October 2013 Accepted 22 October 2013 Available online 8 November 2013 Keywords: Influenza viruses Recombinant proteins Bacteria Antibody library Fusion partner ELISA
Although antiviral drugs and vaccines have been successful for mitigating influenza virus infections, the lack of general technical platform for the timely supply of soluble and highly purified influenza viral antigens presents a serious bottleneck for the subsequent analysis for the effective control of the viral disease. Using the Escherichia coli (E. coli) lysyl tRNA synthetase (LysRS) as a novel fusion partner, this study reports the soluble expression of influenza viral proteins in E. coli host, construction of antibody library against the virus, and detection of anti-influenza antibodies using the expressed viral antigens. When influenza A and B viral proteins were fused with the LysRS, the fusion proteins were expressed predominantly as soluble forms and their production yields were high enough to be amenable to immunization protocols in rabbits for antibody generation. The produced antibodies showed high level binding specificity against the respective viral proteins, with cross-reactivity across heterologous viruses within the same type of influenza viruses. In addition, LysRS-HA fusion protein could bind specifically to anti-HA antibodies in the virus-infected mouse serum in widely accepted two detection methods, Western blot and ELISA. These results present a convenient tool for the production of antibodies specific to the virus as well as the rapid detection of influenza viral infections, ultimately contributing to the control of influenza viruses including highly pathogenic H5N1, pandemic H1N1, or the recent H7N9 influenza viruses. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Influenza virus poses persistently serious threats to human health, which become more pronounced in the event of sporadic pandemics by human-to-human transmission of highly pathogenic viruses (Potter, 2001; Nicholson et al., 2003). While inactivated killed vaccines and cold-adapted live attenuated vaccines can be prepared from cultured influenza viruses, the development of recombinant protein vaccines or in vitro antiviral drug or target screenings need a substantial amount of soluble and highly purified viral antigens. Although Escherichia coli systems remain the most preferred choice for the over-expression of heterologous proteins due to distinct advantages including inexpensive carbon source requirements, rapid growth rates, and the simplicity of scale-up (Baneyx, 1999), influenza viral proteins have been hardly amenable to soluble expression in this host, and, in particular, it has been
∗ Corresponding author at: Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, South Korea. Tel.: +82 2 2123 2885; fax: +82 2 362 7265. E-mail addresses: [email protected], [email protected] (B.L. Seong). 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.10.035
considered extremely difficult to express transmembrane proteins, such as hemagglutinin (HA), neuraminidase (NA), and M2 ion channel protein, as soluble forms. With maltose-binding protein (MBP), N-utilizing substance-A (NusA), and glutathione S-transferase (GST) at the head of list (Smith and Johnson, 1988; Davis et al., 1999; Fox and Waugh, 2003; Nallamsetty and Waugh, 2006), a number of fusion partners have been advanced as solubility enhancers for the bacterial expression of aggregation-prone proteins (Young et al., 2012). However, most of the fusion partners still failed to support soluble expression for target proteins (Esposito and Chatterjee, 2006), underlining the need for a novel fusion partner that has general versatility in broad-spectrum. Recently, it was reported that RNA-binding proteins (RBP) could serve as novel fusion partners that significantly accelerated the folding and solubility of passenger proteins in E. coli (Kim et al., 2007; Choi et al., 2008). Consistent with several reports that RNA had accelerated the folding of interacting proteins (Frankel and Smith, 1998; Rentzeperis et al., 1999; Uversky et al., 2000), interaction with RNA stimulated the folding of the RBPs themselves and also promoted the folding and soluble expression of their fused aggregation-prone proteins (Choi et al., 2008). In addition, the RBP demonstrated superior efficiency in gaining
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and enhancing the solubility to MBP, a widely used fusion partner (Choi et al., 2008), exhibiting a potential to serve as an alternative powerful tool for recombinant protein expression. With a view to generate antibody library against the influenza viruses, the RBP fusion strategy was applied into the soluble expression and purification of influenza internal and surface membrane proteins by fusion to E. coli lysyl tRNA synthetase (LysRS). The prominent ability of the LysRS fusion to induce high yield soluble expression of influenza viral proteins presents significant benefits for the production of viral antigen or antibody libraries for analytical, therapeutic, prophylactic, and diagnostic purposes in numerous non-clinical and clinical investigations. 2. Materials and methods 2.1. Construction of protein expression vectors pGE-LysRS4 vector constructed in previous work (Choi et al., 2008) was used as a vector for the expression of influenza viral proteins in E. coli. The LysRS expression cassette includes LysRS, D6 hexa-aspartic acids linker, TEV recognition site, multicloning site, and the hexa-histidines tag under the T7 promoter (Fig. 1A). The cDNAs each encoding the complete open reading frame (ORF) of individual viral proteins from A/WSN/33 (H1N1), A/Puerto Rico/8/34 (H1N1), and B/Yamagata/16/88 were amplified by reverse transcription PCR (RT-PCR) and then inserted into the pGE-LysRS4 vector. 2.2. Protein expression Protein expression and SDS-PAGE analysis were performed as described previously (Choi et al., 2008). Each expression plasmid was transformed into the E. coli expression host, BL21 (DE3) pLysS. A single colony of transformants was inoculated into 3 ml of LB containing both 50 g/ml ampicillin and 34 g/ml chloramphenicol. The culture medium was then diluted into 25 ml of the fresh LB containing the same antibiotics and the cells were cultured to the optical density (OD) of 0.5 at 600 nm. Expression of proteins was induced for 6 h at 20–30 ◦ C by the addition of 1 mM IPTG. After centrifugation, the cell pellets were suspended in 0.3 ml of PBS and disrupted by sonication. The cell lysates were centrifuged at 11,000 × g for 12 min and separated into soluble and pellet fractions. Each fraction was then mixed with the same volume of 2× SDS loading buffer and boiled. These samples were subjected to SDS-PAGE and visualized by staining with Coomassie brilliant blue R-250.
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bovine serum albumin (BSA) (Amresco, Solon, OH, USA) of a known concentration as control. 2.4. Animal cells and influenza viruses Madin–Darby canine kidney (MDCK) cells were maintained in minimal essential medium supplemented with 10% fetal bovine serum. To evaluate the binding specificities of antisera collected from the rabbits immunized with the fusion proteins, MDCK cells were infected with 1 multiplicity of infection (MOI) of each of four influenza A viruses, A/WSN/33 (H1N1), A/Singapore/6/86 (H1N1), A/Sydney/5/97 (H3N2), A/Puerto Rico/8/34 (H1N1), and of three influenza B viruses; B/Lee/40, B/Yamagata/16/88, and cold-adapted B/Lee/40 (Seo et al., 2008), and the cells were harvested 24 h after the infections for Western blots. 2.5. Immunization and polyclonal antibodies production in rabbits Rabbit experiments were conducted in the animal facilities of the LabFrontier (Anyang, South Korea). To obtain polyclonal antibodies specific to influenza viral proteins, two rabbits were immunized with each of purified LysRS-fused recombinant influenza viral proteins. Each 0.5 mg of LysRS-fused protein emulsified with Freund’s complete adjuvant was injected intradermally at multiple sites on the back of each rabbit. Starting from four weeks after the first immunization, each animal was boosted twice by an additional 0.2 mg of the protein and adjuvant mixtures with the interval of two weeks. Sera samples were collected at seven days after each boosting and the final sera samples were aliquoted and stored at −80 ◦ C. The experimental protocol was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) of LabFrontier. 2.6. Mice infection and serum preparation 6-Week-old female BALB/c mice were infected intranasaly with 106 PFU of attenuated mutant strain of A/Puerto Rico/8/34 (H1N1) virus (Jang et al., 2013) or mock-infected with PBS as a control. Three weeks later the mice blood samples were collected from the mice via retro-orbital bleeding and clotted at 4 ◦ C overnight to collect sera samples. Mouse study was carried out in strict accordance with the recommendations of the Korean Food and Drug Administration (KFDA) guidelines. Protocols were reviewed and approved by the IACUC of the Yonsei Laboratory Animal Research Center (YLARC) (Permit number: 2012-0094). 2.7. ELISA
2.3. Purification and quantification of fusion proteins Proteins were purified from 1 l culture of each transformant using nickel affinity chromatography. The harvested cell pellets were suspended in 5 ml of equilibration buffer A (20 mM Tris–HCl (pH 7.5), 300 mM NaCl, 10% glycerol, 2 mM 2-mercaptoethanol, and 5 mM imidazole) and disrupted by sonication. The soluble fractions were recovered by centrifugation at 11,000 × g for 20 min and then applied onto HiTrap chelating HP column (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK). After washing, proteins were eluted with 50 ml of elution buffer with the same composition of equilibration buffer as above except for the linear gradients of imidazole ranging from 10 mM to 300 mM. The fractions containing proteins of interest were pooled, concentrated, dialyzed against the buffer containing 50 mM Tris–HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, and 1 mM DTT in 50% glycerol. After SDS-PAGE, the purified proteins were quantified by gel densitometry scanning of the Coomassie-blue stained protein band using
Microtiter 96-well plates were coated with 100 ng/well of LysRS-fused influenza viral protein and incubated at 4 ◦ C overnight. The plates were washed and blocked by 1% BSA for 1 h at room temperature (RT). 100 l of sera with various dilutions were added to the wells for 1 h, and the same volume of a secondary goat anti-rabbit IgG antibody or goat-anti mouse IgG antibody conjugated with HRP (Sigma–Aldrich, St. Louis, MO, USA) was treated for 1 h. After washings, substrate solution was added to the well and the plate was incubated in the dark. The colorimetric reaction was stopped by the addition of 100 l/well of 1 M H2 SO4 and the absorbance was read at 492 nm on ELISA reader (FLUOstar OPTIMA, BMG LABTECH, Offenburg, Germany). 2.8. Western blot analysis MDCK cells infected with one MOI of each influenza virus were subjected to Western blot analysis using each final serum
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Fig. 1. Soluble expression of LysRS-fused influenza A or B viral proteins in E. coli. (A) Schematic illustration of pGE-LysRS4 vector used for the expression of fusion proteins in E. coli. (B) Expression profiles of ten fusion proteins in soluble or insoluble fraction. The fusion proteins were expressed at 27 ◦ C (for all proteins except for A-NA) or 20 ◦ C (for A-NA) and their solubility was analyzed by SDS-PAGE. M, T, S, and P represent molecular weight marker in kDa, total lysates, soluble fraction, and insoluble fraction, respectively. Arrows indicate the induced fusion proteins. (C) Solubility of each of fusion proteins in B estimated by gel densitometer.
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produced from immunized rabbits. Various dilutions of each serum were tested in the analysis to determine optimal dilution, which was presented in results. As secondary antibodies, we used goat anti-rabbit IgG monoclonal antibody conjugated with horseradish peroxidase (Sigma–Aldrich, St. Louis, MO, USA). For the detection of anti-HA and anti-NA antibodies in influenza virus-infected mouse sera, 500 ng of LysRS-HA and LysRS-NA fusion proteins were loaded onto SDS-PAGE, which were then subjected to Western blot with the mouse serum with a dilution of 1:500. 3. Results 3.1. Soluble expression of LysRS-fused influenza viral proteins Full-length five viral proteins (including three internal proteins, M1, NS1, and NS2, and two surface membrane proteins NA and M2) of A/WSN/33 (H1N1) virus and HA protein of A/Puerto Rico/8/34 (H1N1) were subcloned into pGE-LysRS4 vector as a fusion to E. coli lysyl tRNA synthetase (LysRS). Similarly, the four proteins (M1, M2, NS1, and NS2) of B/Yamagata/16/88 virus were subcloned into the same expression vector. All fusion proteins were tested for soluble expression in E. coli host, BL21 (DE3) pLysS (Fig. 1A). Protein expression was induced by the addition of 1 mM IPTG under various temperature conditions, and the cell lysates were fractionated into soluble supernatants and insoluble pellets to determine the solubility of the expressed proteins. Through repetitive screenings of temperature and solubility, it was found that the most of the fusion proteins were expressed predominantly as soluble forms at 27 ◦ C. Interestingly, the expression level of full-length HA protein was poor, but truncation of 35 amino acids residue encoding C-terminal transmembrane and cytoplasmic domain dramatically increased the expression level of the HATM protein (Fig. 1B). Overall, the solubility was ≥80% for all proteins expressed (Fig. 1C). The high solubility of fusion proteins at relatively lower temperatures was consistent with previous results that the solubility of RBP-fused proteins increased substantially at lower temperature (Choi et al., 2008). Of note, the surface membrane protein NA of A/WSN/33 (H1N1) virus was most soluble at 20 ◦ C (Fig. 1B). Likewise, transmembrane proteins, A-M2 and B-M2, were also successfully expressed as soluble forms (Fig. 1B). Thus, the LysRS demonstrated prominent ability to induce the soluble expression of its linked protein, regardless of whether the cargo is an internal or transmembrane protein. 3.2. Scale-up and purification of fusion proteins Expressed fusion proteins were purified from 1 l culture of each transformant using one-step nickel chromatography. After cell lysis and fractionation, soluble fractions that contained the most of expressed fusion proteins were applied onto a column and eluted with linear gradients of imidazole ranging 10–300 mM. Hexa-histidines-tagged fusion proteins were purified efficiently from crude extract with high level homogeneity, albeit with the minor mixtures of probably E. coli-derived histidine-rich proteins or degraded fragments from the fusion proteins (Fig. 2A). The SDS-PAGE analysis of protein profiles at different stages of purification step showed that the most of each purified fusion protein migrated with expected molecular mass, indicating that, despite their relatively large molecular size ranging from 72 kDa to 114 kDa, the fusion proteins remained highly soluble during the scale-up expression and subsequent purification steps. The elution fractions containing the target fusion protein were pooled and dialyzed. Each of purified fusion proteins was subjected to SDS-PAGE analysis and quantified by comparing the band density with BSA of known concentration by gel densitometer scanning (Fig. 2B). The final yields of
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the fusion proteins were in the range of 1–2 mg/ml. Taken together, the LysRS showed excellent solubilizing and stabilizing ability during the expression and purification of influenza viral proteins in E. coli. 3.3. Antibody production against influenza viral proteins Among the ten fusion proteins expressed, five fusion proteins derived from influenza A/WSN/33 (H1N1) virus were used for antibody production from rabbits. Rabbits were primarily immunized with each fusion protein, and four weeks later the rabbits were boosted two times in two weeks interval. To estimate the level of specific antibody contained in the final serum, ELISA was performed using the fusion proteins as coated antigens. As compared to pre-immune sera, all five sera samples contained high level antibodies, with strong ELISA response even after 100,000fold dilution (Fig. 3A). The ELISA results may represent antibody responses against either LysRS or viral protein, or both, and thus do not reflect the exact levels of viral protein-specific antibodies. Therefore, Western blot analysis was performed on influenzainfected cells to examine whether the produced antibodies could bind specifically to their respective target viral proteins. MDCK cells were infected with 1 MOI of various influenza A and B virus strains, and the cell lysates were subjected to Western blot analysis to investigate the breadth of specificities of the antibodies. All of the five antibodies bound specifically to the viral proteins of influenza A viruses but not to those of B viruses (Fig. 3B). Although the fusion proteins were derived from H1N1 strain, the antibodies showed cross-reactivity against heterologous H1N1 and H3N2 strains. The cross-reactivity observed in the M1, M2, NS1, and NS2 was reasonably predictable since those proteins were highly conserved within the same type viruses. However, the cross reactivity shown in the NA antibody was remarkable, considering that the sequence homology of the NA proteins between A/WSN/33 (H1N1) and A/Sydney/5/97 (H3N2) was substantially low (41% in identities and 57% in positives). Thus, the antibodies exhibited a wide range of binding specificities within the influenza A type viruses against conserved internal proteins as well as highly variable surface antigen. 3.4. Detection of anti-influenza antibodies in infected mouse using soluble viral proteins Besides the production of antibodies specific to the viral antigens, highly soluble and purified viral proteins can be used for the detection of specific antibodies in virus-infected animals and humans. To explore this potential, the mice were infected with 106 PFU of the attenuated strain of A/Puerto Rico/8/34 (H1N1) (Jang et al., 2013) and the serum samples were tested for the presence of virus-specific antibodies using the two most popular detection tools, Western blot and ELISA. In the Western blot, anti-HA antibodies in all the infected mice sera were detected easily by the LysRS-HATM fusion protein. However, the anti-NA antibodies were barely detectable by the LysRS-NA proteins, with only one of four mice serum generating observable band (Fig. 4A). Likewise, when 100 ng/well of each fusion protein was coated into a microtiter plate, ELISA produced high level detection signal to LysRS-HATM protein in all the vaccinated mice sera applied, whereas the signal to LysRS-NA protein was nearly negative with similar absorbance to the LysRS control and PBS group (Fig. 4B). Increase in the concentration of the LysRS-NA protein did not produce any noticeable difference as compared to the controls in both assays. Considering the efficient production of anti-NA antibodies in the rabbits immunized with LysRS-NA protein (Fig. 3), poor detection ability of the LysRS-NA proteins can be reasonably attributed to much lower abundance of anti-NA antibodies in the
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Fig. 2. Purification of influenza A or B viral fusion proteins. (A) SDS-PAGE analysis showing protein profiles during the purification step. M, protein size marker; L, lysate of soluble fraction; F, unbound flow through; W, washing step. Arrows indicate the target fusion proteins. (B) SDS-PAGE analysis of each of pooled fusion proteins from A. M, protein size marker (kDa). Arrows indicate the induced fusion proteins.
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Fig. 3. Antibody productions against the influenza A viral proteins. (A) The antibodies levels produced in the rabbits immunized with LysRS-fused influenza A viral proteins. Serum IgG antibodies levels in pre-immune or final serum were estimated by ELISA using fusion viral proteins as coated antigens. The result of one of the two immunized rabbits was shown. (B) Binding specificities of antibodies against influenza viral proteins. MDCK cells were infected with 1 MOI of each of influenza A or B viruses, or mock-infected, and the cells were harvested at 24 h after the infections. The cell lysates were subjected to Western blot analysis using each final antiserum with an indicated dilution.
vaccinated mice sera relative to anti-HA antibodies (Kilbourne et al., 1987; Feng et al., 2009). Taken together, these results show that the E. coli-expressed influenza HATM protein can serve as useful detection tools for measuring the quantity or quality of antibody responses to the viruses.
4. Discussion In this study antigen and antibody libraries were established against influenza viruses using E. coli expression system, in which the LysRS was employed as a novel fusion partner to successfully
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Fig. 4. Detection of anti-influenza antibodies in virus-infected mice sera using LysRS-fused viral proteins. (A) Western blot analysis for the detection of anti-influenza surface antigen antibodies. Mice were infected with 106 PFU of mutant strain of A/Puerto Rico/8/34 (H1N1) (n = 4) or mock-infected with PBS (n = 1), and three weeks later sera were collected from the mice. 500 ng of LysRS, LysRS-HATM, and LysRS-NA proteins were run by SDS-PAGE and then subjected to Western blot with the each mouse serum of a dilution of 1:500. () Indicates the band of full-length fusion protein, LysRS-HATM (114 kDa) or LysRS-NA (111 kDa). (B) ELISA for the detection of anti-influenza surface antigen antibodies. Each microtiter 96-well plate was coated with 100 ng/well of LysRS, LysRS-HATM, or LysRS-NA protein, and the twofold serial dilutions of the serum of vaccinated (V, n = 5) or nonvaccinated (P, n = 3) mice were applied onto the plate for the analysis. Scanned images of the plates after the analysis are shown (upper), and their measured optical density (OD) of each mouse serum for each coated antigen protein are shown in the same graph (lower).
express all viral proteins as soluble forms. The specificity of antibodies is consistent with the serological classification of influenza viruses (Krug, 2001), which attests to the quality of antigens produced in this report. Previously, the full-length influenza viral
proteins have been refractory to soluble expression in E. coli host, with potential exception for the NP proteins (Huang et al., 2012). More notably, the bacterial expressions of the transmembrane proteins of influenza virus, such as HA, NA, and M2, showed low
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efficiency even when expressed and recovered from insoluble pellets (Davis et al., 1981; Jones and Brownlee, 1985; Ebrahimi et al., 2010), consequently leading to the changes of expression system to relatively expensive eukaryotic hosts, especially for recombinant vaccines based on the HA or M2 (Treanor et al., 1996; Wei et al., 2008; Bosch et al., 2010; Murugan et al., 2013). As the first report of the soluble expression of full-length influenza viral proteins in E. coli, these results present a generally applicable toolbox for the establishment of antigen and antibody libraries of human viruses and their uses in the detection assays as well. Previous study showed that most of aggregation-prone human proteins tested were expressed as soluble forms when fused with the LysRS, which were much more efficient than MBP fusion (Choi et al., 2008). Robust folding and solubility enhancement is now extended to human-infecting viral proteins as exemplified by the influenza proteins in the present study. Notably, the solubility enhancement was robust also for the transmembrane proteins, e.g., HA, NA, or M2, which usually aggregate via hydrophobic interactions in the membrane spanning region. These data together demonstrate that the present fusion system may offer a high-throughput soluble expression platform of proteins of eukaryotic or viral origin, overcoming technical difficulties still persisting in fusion technologies (Esposito and Chatterjee, 2006). Substantial amount of soluble and purified viral proteins is prerequisite for the generation of specific antibodies as well as highly sensitive detection methods, for which the present strategy met the demand successfully. In addition, the production of soluble viral antigen may provide better opportunity to control the viral diseases by the development of recombinant vaccines or antiviral drug screening in vitro. E. coli expression systems have been used for the production of recombinant influenza vaccines, but limited only to sub-domain of the HA or M2, due to low expression and inclusion body formation of the whole proteins (Neirynck et al., 1999; Ernst et al., 2006; Aguilar-Yanez et al., 2010; Khurana et al., 2010; DuBois et al., 2011; Khurana et al., 2011). On the other hand, the soluble expression of full-length NA and M2 proteins by fusion with the LysRS may be very helpful for developing recombinant vaccine spanning all antigenic epitopes required for sufficient protective efficacy. Influenza viruses exhibit extremely diverse antigenicity comprising various combinations of 16 HA and 9 NA subtypes (Fouchier et al., 2005). The importance of a timely supply of soluble antigens and corresponding antibodies cannot be overstated in the epidemiological studies of influenza viruses, especially considering the zoonotic nature of influenza transmission. The present results therefore could be extended to other influenza strains, including H5N1 highly pathogenic avian influenza (Vijaykrishna et al., 2008), 2009 pandemic H1N1 influenza (Neumann et al., 2009), and the recent 2013 H7N9 strains with high mortality or morbidity among human population (Uyeki and Cox, 2013). While further extending the utility of RNAinteraction mediated protein folding for recombinant proteins (Choi et al., 2009, 2011, 2012), the present results are expected to provide practical means to establish antigen and antibody libraries for infectious agents for analytical, diagnostic, and prophylactic purposes.
Role of the funding source This work was supported by a grant from the Korea Healthcare Technology R&D project of Ministry for Health, Welfare and Family Affairs of Republic of Korea [Grant number A085105]. The funding source did not have roles in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
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