Expression and purification of MERS-CoV envelope protein, an essential viroporin, using the baculovirus expression system
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Abstract
Background and Objectives: The causative agent of Middle East Respiratory Syndrome (MERS) is a zoonotic Coronavirus (MERS-CoV) identified in Saudi Arabia in 2012. The envelope (E) protein of MERS-CoV is a small viral protein which plays several essential roles during virus replication. To facilitate study of the structure and function of the E protein, recombinant MERS-CoV E protein was expressed using the baculovirus expression system.
Materials and Methods: A recombinant E open reading frame including an 8-histidine tag at the amino terminus was designed and cloned into a baculovirus transfer vector. Following construction of a recombinant virus insect cells were infected and the expression of the E protein assessed by SDS-PAGE and Western blotting.
Results: Recombinant E protein, tagged at the N-terminus with a polyhistidine sequence, with a molecular mass of 10.18 kD was identified by Western blotting with an anti-His antibody. Following large scale infection E protein was released by detergent mediated lysis of infected cells and purified by Immobilized Metal Ion Affinity Chromatography (IMAC).
Conclusion: Purified full length recombinant MERS-CoV E protein can be isolated by IMAC and is suitable for further functional, biophysical or immunological studies.
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20. Parthasarathy K, Lu H, Surya W, Vararattanavech A, Pervushin K, Torres J. Expression and purification of coronavirus envelope proteins using a modified β-barrel construct. Protein Expr Purif 2012; 85: 133-141.
21. Shen X, Xue J-H, Yu C-Y, Luo H-B, Qin L, Yu X-J, et al. Small envelope protein E of SARS: cloning, expression, purification, CD determination, and bioinformatics analysis. Acta Pharmacol Sin 2003; 24: 505-511.
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25. Chen W-S, Villaflores OB, Jinn T-R, Chan M-T, Chang Y-C, Wu T-Y. Expression of recombinant human interferon-γ with antiviral activity in the Bi-cistronic baculovirus-insect/larval system. Biosci Biotechnol Biochem 2011; 75: 1342-1348.
26. Gao J, Lu G, Qi J, Li Y, Wu Y, Deng Y, et al. Structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the S protein of Middle East respiratory syndrome coronavirus. J Virol 2013; 87: 13134-13140.
27. Kitts PA, Ayres MD, Possee RD. Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors. Nucleic Acids Res 1990; 18: 5667-5672.
28. Hitchman RB, Possee RD, King LA. Baculovirus expression systems for recombinant protein production in insect cells. Recent Pat Biotechnol 2009; 3: 46-54.
2. Neuman BW, Buchmeier MJ. Supramolecular architecture of the Coronavirus particle. Adv Virus Res 2016; 96: 1-27.
3. Calisher C, Carroll D, Colwell R, Corley RB, Daszak P, Drosten C, et al. Statement in support of the scientists, public health professionals, and medical professionals of China combatting COVID-19. Lancet 2020; 395(10226): e42-e43.
4. Ou X, Liu Y, Lei X, Li P, Mi D, Ren L, et al. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat Commun 2020; 11: 1620.
5. Van Boheemen S, De Graaf M, Lauber C, Bestebroer TM, Raj VS, Zaki M, et al. Genomic characterization of newly discovered Coronavirus associated with acute respiratory distress syndrome in humans. mBio 2012; 3(6): e00473-12.
6. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus ADME, Fouchier RAM. Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia. N Engl J Med 2012; 367: 1814-1820.
7. Mohd HA, Al-Tawfiq JA, Memish ZA. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) origin and animal reservoir. Virol J 2016; 13: 87.
8. Park J-E, Jung S, Kim A, Park J-E. MERS transmission and risk factors: A systematic review. BMC Public Health 2018; 18: 574.
9. Matsuyama R, Nishiura H, Kutsuna S, Hayakawa K, Ohmagari N. Clinical determinants of the severity of Middle East respiratory syndrome (MERS): A systematic review and meta-analysis. BMC Public Health 2016; 16: 1203.
10. Hui DS, Azhar EI, Kim Y-J, Memish ZA, Oh M-D, Zumla A. Middle East respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission. Lancet Infect Dis 2018; 18(8): e217-e227.
11. Azhar EI, El-kafrawy SA, Farraj SA, Hassan AM, Al-saeed MS, Hashem AM, et al. Evidence for camel-to-human transmission of MERS Coronavirus. N Engl J Med 2014; 370: 2499-2505.
12. Alsalihi SF, Abdelkadhim Jawad A, Al-Rodhan MA. Molecular study and phylogenetic analysis of middle east respiratory syndrome Corona Virus (MERSCoV) in camel and human. J Phys Conf Ser 2019; 1294: 062097.
13. Raamsman MJ, Locker JK, de Hooge A, de Vries AA, Griffiths G, Vennema H, et al. Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E. J Virol 2000; 74: 2333-2342.
14. Liao Y, Lescar J, Tam JP, Liu DX. Expression of SARS-coronavirus envelope protein in Escherichia coli cells alters membrane permeability. Biochem Biophys Res Commun 2004; 325: 374-380.
15. Madan V, Garcia Mde J, Sanz MA, Carrasco L. Viroporin activity of murine hepatitis virus E protein. FEBS Lett 2005; 579: 3607-3612.
16. Surya W, Li Y, Verdià-bàguena C, Aguilella VM, Torres J. MERS coronavirus envelope protein has a single transmembrane domain that forms pentameric ion channels. Virus Res 2015; 201: 61-66.
17. DeDiego ML, Álvarez E, Almazán F, Rejas MT, Lamirande E, Roberts A, et al. A severe acute respiratory syndrome Coronavirus that lacks the E Gene is attenuated in vitro and in vivo. J Virol 2007; 81: 1701-1713.
18. Targovnik AM, Simonin JA, Mc Callum GJ, Smith I, Cuccovia Warlet FU, Nugnes MV, et al. Solutions against emerging infectious and noninfectious human diseases through the application of baculovirus technologies. Appl Microbiol Biotechnol 2021; 105: 8195-8226.
19. Alsaadi EAJ, Neuman BW, Jones IM. Identification of a membrane binding peptide in the envelope protein of MHV coronavirus. Viruses 2020; 12: 1054.
20. Parthasarathy K, Lu H, Surya W, Vararattanavech A, Pervushin K, Torres J. Expression and purification of coronavirus envelope proteins using a modified β-barrel construct. Protein Expr Purif 2012; 85: 133-141.
21. Shen X, Xue J-H, Yu C-Y, Luo H-B, Qin L, Yu X-J, et al. Small envelope protein E of SARS: cloning, expression, purification, CD determination, and bioinformatics analysis. Acta Pharmacol Sin 2003; 24: 505-511.
22. Álvarez E, DeDiego ML, Nieto-Torres JL, Jiménez-Guardeño JM, Marcos-Villar L, Enjuanes L. The envelope protein of severe acute respiratory syndrome coronavirus interacts with the non-structural protein 3 and is ubiquitinated. Virology 2010; 402: 281-291.
23. Ahrens U, Kaden V, Drexler C, Visser N. Efficacy of the classical swine fever (CSF) marker vaccine Porcilis® Pesti in pregnant sows. Vet Microbiol 2000; 77: 83-97.
24. Yousefi A, Fotouhi F, Hosseinzadeh S, Kheiri MT, Farahmand B, Montazeri S, et al. Expression of antigenic determinants of the haemagglutinin large subunit of novel influenza virus in insect cells. Folia Biol (Praha) 2012; 58: 151-156.
25. Chen W-S, Villaflores OB, Jinn T-R, Chan M-T, Chang Y-C, Wu T-Y. Expression of recombinant human interferon-γ with antiviral activity in the Bi-cistronic baculovirus-insect/larval system. Biosci Biotechnol Biochem 2011; 75: 1342-1348.
26. Gao J, Lu G, Qi J, Li Y, Wu Y, Deng Y, et al. Structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the S protein of Middle East respiratory syndrome coronavirus. J Virol 2013; 87: 13134-13140.
27. Kitts PA, Ayres MD, Possee RD. Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors. Nucleic Acids Res 1990; 18: 5667-5672.
28. Hitchman RB, Possee RD, King LA. Baculovirus expression systems for recombinant protein production in insect cells. Recent Pat Biotechnol 2009; 3: 46-54.
| Files | ||
| Issue | Vol 15 No 1 (2023) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v15i1.11926 | |
| Keywords | ||
| Middle east respiratory syndrome; Coronaviruses; MERS-CoV; Envelope protein; Baculovirus; Insect cells; Immobilised metal-affinity chromatography | ||
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Alsaadi E, Alghezi D, Jones I. Expression and purification of MERS-CoV envelope protein, an essential viroporin, using the baculovirus expression system. Iran J Microbiol. 2023;15(1):121-127.
