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Generation of a fusion protein of the extracellular domain of BR3 with the Fc fragment of human IgG1 (sBR3-Fc) in Pichia pastoris as an antagonist for BLyS

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Abstract

Elevated levels of B cell-activating factor of the TNF family (BlyS) have been implicated in the pathogenesis of autoimmune diseases in human. Removal of pathogenic B lymphocytes by decoy receptors has demonstrated clinical benefit in both oncological and immunological diseases. In this report, we have constructed vectors for the convenient and rapid expression of the extracellular domain of BR3(sBR3) fused to the Fc fragment (hinge, CH2, CH3) of human IgG1 in the methylotrophic yeast, Pichia pastoris. SDS-PAGE assays of culture broth from a methanol-induced expression strain demonstrated that the recombinant sBR3-Fc fusion protein is secreted and recovered from the culture medium as a disulfide-linked, glycosylated homodimer. The recombinant protein was purified to >95% using protein A affinity chromatography and size exclusion chromatography steps. Bioactivity of the recombinant sBR3-Fc was confirmed by the ability of the protein to inhibit mouse B lymphocyte proliferation induced by BLyS in vitro. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional sBR3-Fc fusion protein for both research and industrial purposes.

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References

  1. Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326

  2. Gross JA, Johnston J, Mudri S, Enselman R, Dillon SR, Madden K, Xu W, Parrish-Novak J, Foster D, Lofton-Day C, Moore M, Littau A, Grossman A, Haugen H, Foley K, Blumberg H, Harrison K, Kindsvogel W, Clegg CH (2000) TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature 404:995–999

  3. Kalled SL (2006) Impact of the BAFF/BR3 axis on B cell survival, germinal center maintenance and antibody production. Semin Immunol 18(5):290–296

  4. Laemmli UK (1970) Cleavage of structural proteins during assembly of the heat of bacteriophage T4. Nature 277:680–685

  5. Lee CV, Hymowitz SG, Wallweber HJ, Gordon NC, Billeci KL, Tsai SP, Compaan DM, Yin J, Gong Q, Kelley RF, DeForge LE, Martin F, Starovasnik MA, Fuh G (2006) Synthetic anti-BR3 antibodies that mimic BAFF binding and target both human and murine B cells. Blood 108(9):3103–3111

  6. Mantchev GT, Cortesao CS, Rebrovich M, Cascalho M, Bram RJ (2007) TACI is required for efficient plasma cell differentiation in response to T-independent type 2 antigens. J Immunol 179:2282–2288

  7. Moore PA, Belvedere O, Orr A, Pieri K, LaFleur DW, Feng P, Soppet D, Charters M, Gentz R, Parmelee D, Li Y, Galperina O, Giri J, Roschke V, Nardelli B, Carrell J, Sosnovtseva S, Greenfield W, Ruben SM, Olsen HS, Fikes J, Hilbert DM (1999) BLyS: member of the tumor necrosis factor family and B lymphocyte stimulator. Science 285:260–263

  8. Mukhopadhyay A, Ni J, Zhai Y, Yu GL, Aggarwal BB (1999) Identification and characterization of a novel cytokine, THANK, a TNF homologue that activates apoptosis, nuclear factor-κB, and c-Jun NH2-terminal kinase. J Biol Chem 274:15978–15981

  9. Nardelli B, Belvedere O, Roschke V, Moore PA, Olsen HS, Migone TS, Sosnovtseva S, Carrell JA, Feng P, Giri JG, Hilbert DM (2001) Synthesis and release of B-lymphocyte stimulator from myeloid cells. Blood 97:198–204

  10. Nimmanapalli R, Lyu MA, Du M, Keating MJ, Rosenblum MG, Gandhi V (2007) The growth factor fusion construct containing B-lymphocyte stimulator (BLyS) and the toxin rGel induces apoptosis specifically in BAFF-R-positive CLL cells. Blood 15:2557–2564

  11. O'Connor BP, Raman VS, Erickson LD, Cook WJ, Weaver LK, Ahonen C, Lin LL, Mantchev GT, Bram RJ, Noelle RJ (2004) BCMA is essential for the survival of long-lived bone marrow plasma cells. J Exp Med 199:91–98

  12. Rodig SJ, Shahsafaei A, Li B, Mackay CR, Dorfman DM (2005) BAFF-R, the major B cell-activating factor receptor, is expressed on most mature B cells and B-cell lymphoproliferative disorders. Human Pathol 36(10):1113–1119

  13. Schneider P, MacKay F, Steiner V, Hofmann K, Bodmer JL, Holler N, Ambrose C, Lawton P, Bixler S, Acha-Orbea H, Valmori D, Romero P, Werner-Favre C, Zubler RH, Browning JL, Tschopp J (1999) BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J Exp Med 89:1747–1756

  14. Shu HB, Hu WH, Johnson H (1999) TALL-1 is a novel member of the TNF family that is down-regulated by mitogens. J Leukoc Biol 65:680–683

  15. Thompson JS, Bixler SA, Qian F, Vora K, Scott ML, Cachero TG, Hession C, Schneider P, Sizing ID, Mullen C, Strauch K, Zafari M, Benjamin CD, Tschopp J, Browning JL, Ambrose C (2001) BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF. Science 293:2108–2111

  16. Tribouley C, Wallroth M, Chan V, Paliard X, Fang E, Lamson G, Pot D, Escobedo J, Williams LT (1999) Characterization of a new member of the TNF family expressed on antigen presenting cells. Biol Chem 380:1443–1447

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Acknowledgements

This work was partly supported by grants from the Program of National Science Foundation of China (No. 30701098) and the International Cooperation Program of Jiangsu Province (No. BZ2007078).

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Correspondence to Shuangquan Zhang.

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Cao, P., Zhang, S., Fang, Z. et al. Generation of a fusion protein of the extracellular domain of BR3 with the Fc fragment of human IgG1 (sBR3-Fc) in Pichia pastoris as an antagonist for BLyS. Appl Microbiol Biotechnol 78, 275–282 (2008). https://doi.org/10.1007/s00253-007-1299-8

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Keywords

  • BR3
  • IgG1 Fc
  • Fusion protein
  • Pichia pastoris
  • Secretion expression
  • Protein purification