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

  • cc icon
  • 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.


  • KEYWORD

    PRRSV , baculovirus , SUMO , SUMOstar , Bombyx mori

  • 1. Bautista EM, Molitor TW (1997) T cell responses to the structural polypeptides of porcine reproductive and respiratory syndrome virus. [Arch Virol] Vol.144 P.117-134 google doi
  • 2. Butt TR, Edavettal SC, Hall JP, Mattern MR (2005) SUMO fusion technology for difficult-to-express proteins. [Protein Expr Purif] Vol.43 P.1-9 google doi
  • 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] Vol.193 P.329-339 google doi
  • 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] Vol.329 P.469-478 google doi
  • 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] Vol.30 P.2874-2880 google
  • 6. Indik S, Valicek L, Klein D, Klanova J (2000) Variations in the major envelope glycoprotein GP5 of Czech strains of porcine reproductive and respiratory syndrome virus [J Gen Virol] Vol.81 P.2497-2502 google doi
  • 7. 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] Vol.23 P.1809-1817 google doi
  • 8. Jentsch S, Pyrowolakis G (2000) Ubiquitin and its kin: how close are the family ties? [Trends Cell Biol] Vol.10 P.335-342 google doi
  • 9. Johnson ES (2004) Protein modification by SUMO. [Annu Rev Biochem] Vol.73 P.355-382 google doi
  • 10. 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] Vol.88 P.424-433 google
  • 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] Vol.14 P.107-117 google doi
  • 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] Vol.46 P.709-719 google doi
  • 13. Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. [Nat Biotechnol] Vol.23 P.567-575 google doi
  • 14. Lesley SA (2001) High-throughput proteomics: protein expression and purification in the postgenomic world. [Protein Expr Purif] Vol.22 P.159-164 google doi
  • 15. Li SJ, Hochstrasser M (1999) A new protease required for cell-cycle progression in yeast. [Nature] Vol.398 P.246-251 google doi
  • 16. Li SJ, Hochstrasser M (2000) The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein. [Mol Cell Biol] Vol.20 P.2367-2377 google doi
  • 17. 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] Vol.62 P.21-28 google doi
  • 18. 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] Vol.5 P.75-86 google doi
  • 19. 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] Vol.15 P.182-189 google doi
  • 20. Melchior F (2000) SUMO-nonclassical ubiquitin. [Annu Rev Cell Dev Biol] Vol.16 P.591-626 google doi
  • 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] Vol.55 P.197-202 google doi
  • 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] Vol.71 P.6061-6067 google
  • 23. Muller S, Hoege C, Pyrowolakis G, Jentsch S (2001) SUMO, ubiquitin’s mysterious cousin. [Nat Rev Mol Cell Biol] Vol.2 P.202-210 google doi
  • 24. #8217;Reilly DR, Miller LK, Luckow VA (1992) Baculovirus expression vectors: a laboratory manual. google
  • 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] Vol.13 P.785-792 google doi
  • 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] Vol.78 P.495-502 google doi
  • 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] Vol.21 P.192-200 google doi
  • 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] Vol.42 P.68-74 google doi
  • 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] Vol.579 P.3369-3375 google doi
  • 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] Vol.42 P.100-110 google doi
  • [Fig. 1.] Construction of the recombinant viruses (A) and expression of the PRRSV structural protein in Bm5 cells (B). The cells were infected at an MOI of 5 pfu per cell with the wild type BmNPV or rBmRSV-ORF4, rBmRSV-ORF5, and rBmRSV-ORF6 and harvested at 3 d postinfection. Proteins were separated on a 12% SDS-PAGE (a), transferred to nitrocellulose membranes for Western blot analysis and reacted with anti-6xHis tag (b) and porcine anti-PRRSV antibodies (c). M, protein size marker; Mock, mock-infected cells and BmNPV, wild type BmNPV. The recombinant proteins are indicated with arrows.
    Construction of the recombinant viruses (A) and expression of the PRRSV structural protein in Bm5 cells (B). The cells were infected at an MOI of 5 pfu per cell with the wild type BmNPV or rBmRSV-ORF4, rBmRSV-ORF5, and rBmRSV-ORF6 and harvested at 3 d postinfection. Proteins were separated on a 12% SDS-PAGE (a), transferred to nitrocellulose membranes for Western blot analysis and reacted with anti-6xHis tag (b) and porcine anti-PRRSV antibodies (c). M, protein size marker; Mock, mock-infected cells and BmNPV, wild type BmNPV. The recombinant proteins are indicated with arrows.
  • [Fig. 2.] Construction of the expression plasmid with SUMO fusion (A) and expression of the PRRSV structural protein in Bm5 cells (B). The cells were infected in the absence or presence of 5 μg/mL of tunicamycin with the wild type BmNPV or rBmRSV-ORF4S, rBmRSV-ORF5S, and rBmRSV-ORF6S and harvested at 3 d post-infection. Proteins were separated on a 12% SDS-PAGE (a), transferred to nitrocellulose membranes for Western blot analysis and reacted with 6xHis tag (b) and porcine anti-PRRSV antibodies (c). M, protein size marker and BmNPV, wild type BmNPV. The recombinant proteins and the cleaved SUMO proteins are indicated with arrows.
    Construction of the expression plasmid with SUMO fusion (A) and expression of the PRRSV structural protein in Bm5 cells (B). The cells were infected in the absence or presence of 5 μg/mL of tunicamycin with the wild type BmNPV or rBmRSV-ORF4S, rBmRSV-ORF5S, and rBmRSV-ORF6S and harvested at 3 d post-infection. Proteins were separated on a 12% SDS-PAGE (a), transferred to nitrocellulose membranes for Western blot analysis and reacted with 6xHis tag (b) and porcine anti-PRRSV antibodies (c). M, protein size marker and BmNPV, wild type BmNPV. The recombinant proteins and the cleaved SUMO proteins are indicated with arrows.
  • [Fig. 3.] Expression of the SUMO fusion protein in the fat body of B. mori larvae. Individual silkworm larvae on the first day of the 5th instar were injected with the wild type BmNPV or rBmRSV-ORF4S, rBmRSV-ORF5S, and rBmRSV-ORF6S. At 3 to 5 d post-injection, the fat bodies were collected by dissection and homogenized in lysis buffer. Proteins were separated on a 12% SDS-PAGE (A), transferred to nitrocellulose membranes for Western blot analysis and reacted with 6xHis tag (B) and porcine anti-PRRSV antibodies (C). M, protein size marker; Mock, mock-infected cells and BmNPV, wild type BmNPV. The recombinant proteins are indicated with arrows.
    Expression of the SUMO fusion protein in the fat body of B. mori larvae. Individual silkworm larvae on the first day of the 5th instar were injected with the wild type BmNPV or rBmRSV-ORF4S, rBmRSV-ORF5S, and rBmRSV-ORF6S. At 3 to 5 d post-injection, the fat bodies were collected by dissection and homogenized in lysis buffer. Proteins were separated on a 12% SDS-PAGE (A), transferred to nitrocellulose membranes for Western blot analysis and reacted with 6xHis tag (B) and porcine anti-PRRSV antibodies (C). M, protein size marker; Mock, mock-infected cells and BmNPV, wild type BmNPV. The recombinant proteins are indicated with arrows.
  • [Fig. 4.] Purification of the SUMO fusion protein in the fat body of B. mori larvae. 6xHis-tagged fusion protein was purified from rBmRSV-ORF4S, rBmRSV-ORF5S, and rBmRSV-ORF6S-injected B. mori larvae using the Ni-NTA spin kit under denaturing conditions. Proteins were separated on a 12% SDS-PAGE (A), transferred to nitrocellulose membranes for Western blot analysis and reacted with 6xHis tag (B). M, protein size marker. The recombinant proteins are indicated with arrows.
    Purification of the SUMO fusion protein in the fat body of B. mori larvae. 6xHis-tagged fusion protein was purified from rBmRSV-ORF4S, rBmRSV-ORF5S, and rBmRSV-ORF6S-injected B. mori larvae using the Ni-NTA spin kit under denaturing conditions. Proteins were separated on a 12% SDS-PAGE (A), transferred to nitrocellulose membranes for Western blot analysis and reacted with 6xHis tag (B). M, protein size marker. The recombinant proteins are indicated with arrows.
  • [Fig. 5.] Expression of the SUMOstar fusion protein in the fat body of B. mori larvae. Individual silkworm larvae on the first day of the 5th instar were injected with the wild type BmNPV or rBmRSV-ORF5S*. At 3 to 5 d post-injection, the fat bodies were collected by dissection and homogenized in lysis buffer. Proteins were separated on a 12% SDS-PAGE (A), transferred to nitrocellulose membranes for Western blot analysis and reacted with 6xHis tag (B) and porcine anti-PRRSV antibodies (C). M, protein size marker and BmNPV, wild type BmNPV. The recombinant proteins are indicated with arrows.
    Expression of the SUMOstar fusion protein in the fat body of B. mori larvae. Individual silkworm larvae on the first day of the 5th instar were injected with the wild type BmNPV or rBmRSV-ORF5S*. At 3 to 5 d post-injection, the fat bodies were collected by dissection and homogenized in lysis buffer. Proteins were separated on a 12% SDS-PAGE (A), transferred to nitrocellulose membranes for Western blot analysis and reacted with 6xHis tag (B) and porcine anti-PRRSV antibodies (C). M, protein size marker and BmNPV, wild type BmNPV. The recombinant proteins are indicated with arrows.