DOI QR코드

DOI QR Code

Enhanced expression of the structural protein of porcine reproductive and respiratory syndrome virus (PRRSV) by SUMO fusion

Koo, Hyun Na;Bae, Sung Min;Woo, Soo Dong

  • Received : 2016.06.23
  • Accepted : 2016.06.27
  • Published : 2016.06.30

Abstract

The major structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) are derived from ORFs 4, 5, and 6. They have been considered very important to arouse the humoral and cellular immune responses against PRRSV infection and proposed to be the excellent candidate proteins in the design of PRRS bioengineering vaccine. However, the PRRSV structural proteins are produced in low levels in the infected cells because it forms insoluble protein and possesses several transmembrane regions. To overcome this problem, we fused the ORF4, ORF5, and ORF6 with SUMO (small ubiquitin-related modifier). The resulting fusion protein SUMO-ORF4, -ORF5, and -ORF6 were highly expressed in Bm5 cells. The level of protein expression using the Bombyx mori larvae was higher than that using Bm5 cells. In addition, fusion to SUMOstar, which is not processed by native SUMO proteases, significantly enhanced protein expression levels compared to SUMO fusion. This study demonstrated that SUMO or SUMOstar, when fused with PRRSV structural proteins, was able to promote its soluble expression. This may be a better method to produce PRRSV structural proteins for vaccine development.

Keywords

PRRSV;baculovirus;SUMO;SUMOstar;Bombyx mori

References

  1. Bautista EM, Molitor TW (1997) T cell responses to the structural polypeptides of porcine reproductive and respiratory syndrome virus. Arch Virol 144, 117-134. https://doi.org/10.1007/s007050050489
  2. Butt TR, Edavettal SC, Hall JP, Mattern MR (2005) SUMO fusion technology for difficult-to-express proteins. Protein Expr Purif 43, 1-9. https://doi.org/10.1016/j.pep.2005.03.016
  3. Conzelmann KK, Visser N, van Woensel P, Thiel HJ (1993) Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group. Virology, 193, 329-339. https://doi.org/10.1006/viro.1993.1129
  4. Huang Y, Su Z, Li Y, Zhang Q, Cui L, Su Y, Ding C, Zhang M, Feng C, Tan Y (2009) Expression and Purification of glutathione transferase-small ubiquitin-related modifier-metallothionein fusion protein and its neuronal and hepatic protection against D-galactose-induced oxidative damage in mouse model. J Pharmacol Exp Ther 329, 469-478. https://doi.org/10.1124/jpet.108.149401
  5. Hummel KB, Erdman DD, Heath J, Bellini WJ (1992) Baculovirus expression of the nucleoprotein gene of measles virus and utility of the recombinant protein in diagnostic enzyme immunoassays. J Clin Microbiol 30, 2874-2880.
  6. Indik S, Valícek L, Klein D, Klánová J (2000) Variations in the major envelope glycoprotein GP5 of Czech strains of porcine reproductive and respiratory syndrome virus. J Gen Virol 81, 2497-2502. https://doi.org/10.1099/0022-1317-81-10-2497
  7. Kato T, Murata T, Usui T, Park EY (2004) Improvement of the production of GFPuv-β1,3-N-acetylglucosaminyltransferase 2 fusion protein using a molecular chaperone-assisted insect-cell-based expression system. Biotechnol Bioeng 88, 424-433.
  8. Je YH, Chang JH, Kim MH, Roh JY, Jin BR, O’Reilly DR (2001) The use of defective Bombyx mori nucleopolyhedrovirus genomes maintained in Escherichia coli for the rapid generation of occlusion-postitive and occlusion-negative expression vectors. Biotechnol Lett 23, 1809-1817. https://doi.org/10.1023/A:1012475004212
  9. Jentsch S, Pyrowolakis G (2000) Ubiquitin and its kin: how close are the family ties? Trends Cell Biol 10, 335-342. https://doi.org/10.1016/S0962-8924(00)01785-2
  10. Johnson ES (2004) Protein modification by SUMO. Annu Rev Biochem 73, 355-382. https://doi.org/10.1146/annurev.biochem.73.011303.074118
  11. Koo HN, Bae SM, Choi JB, Shin TY, Yun BNR, Choi JY, Lee KS, Roh JY, Je YH, Jin BR, Yoo SS, Kim JS, Kim YI, Yoon IJ, Woo SD (2011) Characterization and expression of the pseudorabies virus (NYJ strain) glycoproteins in Bombyx mori cells and larvae. J Asia-Pacific Entomol 14, 107-117. https://doi.org/10.1016/j.aspen.2010.08.002
  12. Koo HN, Oh JM, Lee JK, Choi JY, Lee KS, Roh JY, Je YH, Jin BR, Yoo SS, Kim JS, Kim YI, Yoon IJ, Woo SD (2008) Molecular characterization of ORFs 2 to 7 of Korean porcine reproductive and respiratory syndrome virus (CA) and its protein expression by recombinant baculoviruses. J Microbiol 46, 709-719. https://doi.org/10.1007/s12275-008-0224-x
  13. Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23, 567-575. https://doi.org/10.1038/nbt1095
  14. Lesley SA (2001) High-throughput proteomics: protein expression and purification in the postgenomic world. Protein Expr Purif 22, 159-164. https://doi.org/10.1006/prep.2001.1465
  15. Li SJ, Hochstrasser M (1999) A new protease required for cell-cycle progression in yeast. Nature 398, 246-251. https://doi.org/10.1038/18457
  16. Marblestone JG, Edavettal SC, Lim Y, Lim P, Zuo X, Butt TR (2006) Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci 15, 182-189. https://doi.org/10.1110/ps.051812706
  17. Li SJ, Hochstrasser M (2000) The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein. Mol Cell Biol 20, 2367-2377. https://doi.org/10.1128/MCB.20.7.2367-2377.2000
  18. Liu L, Spurrier J, Butt TR, Strickler JE (2008) Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions. Protein Expr Purif 62, 21-28. https://doi.org/10.1016/j.pep.2008.07.010
  19. Malakhov MP, Malakhova OA, Drinker M, Weeks S, Butt TR (2004) SUMO fusion and SUMO-specific proteases for efficient expression and puriWcation of proteins. J Struct Funct Genom 5, 75-86. https://doi.org/10.1023/B:JSFG.0000029237.70316.52
  20. Melchior F (2000) SUMO-nonclassical ubiquitin. Annu Rev Cell Dev Biol 16, 591-626. https://doi.org/10.1146/annurev.cellbio.16.1.591
  21. Meulenberg JJ, Petersen den Besten A, de Kluyver E, van Nieuwstadt A, Wensvoort G, Moormann RJ (1997) Molecular characterization of Lelystad virus. Vet Microbiol 55, 197-202. https://doi.org/10.1016/S0378-1135(96)01335-1
  22. Meulenberg JJ, van Nieuwstadt AP, Essen-Zandbergen A, Langeveld JP (1997) Posttranslational processing and identification of a neutralization domain of the GP4 protein encoded by ORF4 of Lelystad virus. J Virol 71, 6061-6067.
  23. Muller S, Hoege C, Pyrowolakis G, Jentsch S (2001) SUMO, ubiquitin’s mysterious cousin. Nat Rev Mol Cell Biol 2, 202-210. https://doi.org/10.1038/35056591
  24. O’Reilly DR, Miller LK, Luckow VA (1992) Baculovirus expression vectors: a laboratory manual. WH Freeman & Co, New York.
  25. Su Z, Huang Y, Zhou Q, Wu Z, Wu X, Zheng Q, Ding C, Li X (2006) High-level expression and purification of human epidermal growth factor with SUMO fusion in Escherichia coli. Protein Pept Lett 13, 785-792. https://doi.org/10.2174/092986606777841280
  26. Sun Z, Xia Z, Bi F, Liu JN (2008) Expression and purification of human urodilatin by small ubiquitin-related modifier fusion in Escherichia coli. Appl Microbiol Biotechnol 78, 495-502. https://doi.org/10.1007/s00253-007-1330-0
  27. Wu X, Kamei K, Sato H, Sato SI, Takano R, Ichida M, Mori H, Hara S (2001) High-level expression of human acidic fibroblast growth factor and basic fibroblast growth factor in silkworm (Bombyx mori L.) using recombinant baculovirus. Protein Expr Purif 21, 192-200. https://doi.org/10.1006/prep.2000.1358
  28. Wu X, Nie C, Huang Z, Nie Y, Yan Q, Xiao Y, Su Z, Huang Y, Xiao J, Zeng Y (2009) Expression and purification of human keratinocyte growth factor 2 by fusion with SUMO. Mol Biotechnol 42, 68-74. https://doi.org/10.1007/s12033-008-9135-7
  29. Zhou F, Xue Y, Lu H, Chen G, Yao X (2005) A genome-wide analysis of sumoylation-related biological processes and functions in human nucleus. FEBS Lett 579, 3369-3375. https://doi.org/10.1016/j.febslet.2005.04.076
  30. Zuo X, Mattern MR, Tan R, Li S, Hall J, Sterner DE, Shoo J, Tran H, Lim P, Sarafianos SG (2005) Expression and purification of SARS coronavirus proteins using SUMO-fusions. Protein Expr Purif 42, 100-110. https://doi.org/10.1016/j.pep.2005.02.004