Expression and Purification of Brucella spp. Lumazine Synthase Decameric Carrier in Fusion to Extracellular Domain of Influenza M2E Protein

Document Type : Research Article

Authors

1 Education and Extension Organization (AREEO), Agricultural Research, Razi Vaccine and Serum Research Institute, Mashhad Branch, Mashhad, I.R. IRAN

2 Department of biology, Payam Noor university, Tehran, I.R. IRAN

Abstract

Brucella spp. Lumazine synthase enzyme is a decameric protein carrier that displays foreign antigens effectively in a polyvalent manner. The applied strategy using this molecule results in a higher density of antigens and enhances the immunogenicity of peptide vaccines. In the current study, Brucella lumazine synthase (BLS) was applied for fusion with influenza matrix protein 2 ectodomain (M2E) as a foreign peptide. The primary studies were based on bioinformatics tools and the fusion was expressed and purified in the following levels. Forming of the decamer was confirmed by electrophoresis and western blotting techniques. Influenza matrix protein 2 was stably expressed at the 10 amino terminals of lumazine synthase. The purified fusion was injected into mice and immune responses were evaluated with the indirect enzyme-linked immunosorbent assay (ELISA) technique. According to ELISA results yield of the purification process was 41% with the ion-exchange method and the protein was as a single band in Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE). The titer of immunized mice serum with a decameric fusion of lumazine synthase and matrix protein (M2BL) was determined to be more than 1:32000 by indirect ELISA. The level of responses against matrix protein in the decameric state of M2BL, was about 20% higher than monomer M2BL. Anti M2BL was cross-reacted effectively with influenza M2E and in comparison with samples injected with adjuvant, the level of antiM2E was similar. The results in this study confirm the role of multi-copy presentation systems and the applicability of BLS as an antigen carrier and adjuvant in designing peptide vaccines.

Keywords

Main Subjects


[1] Skwarczynski M., Toth I., Peptide-Based Synthetic Vaccines, Chemical Science, 7(2): 842-854 (2016).
[2] Sciutto E., Toledo A., Cruz C., Rosas G., Meneses G., Laplagne D., Ainciart N., Cervantes J., Fragoso G., Goldbaum F.A., Brucella spp. Lumazine Synthase: a Novel Antigen Delivery System. Vaccine, 23(21): 2784-2790 (2005).
[3] López-Sagaseta J., Malito E., Rappuoli R., Bottom Ley M.J., Self-assembling Protein Nanoparticles in the Design of Vaccines, Computational and Structural Biotechnology Journal, 14: 58-68 (2016).
[4] Azuma Y., Edwardson T.G.W., Hilvert D., Tailoring Lumazine Synthase Assemblies for Bionanotechnology, Chem Soc Rev., 47(10): 3543-3557 (2018).
[5] Rossi A.H., Farias A., Fernandez J.E., Bonomi H.R., Goldbaum F.A., Berguer P.M., Brucella spp. Lumazine Synthase Induces a TLR4-Mediated Protective Response Against B16 Melanoma in Mice, PloS one, 10(5):    -     (2015).
[6] Deng L., Cho K.J., Fiers W., Saelens X., M2e-Based Universal Influenza a Vaccines, Vaccines, 3(1): 105-136 (2015).
[7] Cho K.J., Schepens B., Seok J.H., Kim S., Roose K., Lee J-H., Gollardo R., Hamme E.V., Schymowitz J., Rousseau F., Fiers W., Saelens X., Kim K.H.,  Structure of the Extracellular Domain of Matrix Protein 2 of Influenza a Virus in Complex with
a Protective Monoclonal Antibody
, Journal of Virology, 89(7): 3700-3711 (2015).
[8] Majidi B., Najafi M.F., Saeedian S., Cloning, Expression and Purification of Brucella Lumazine Synthase Protein in E. Coli BL21, Journal of Advanced Pharmacy Education & Research| Apr-Jun, 9(S2): 145-149 (2019).
[9] Mejias M.P., Cabrera G., Fernandez-Brando R.J., Baschkier A., Ghersi G., Abrey-Recalde M.J., Miliwebsky E., Meiss R., Goldbaum F., Zylberman V., Rivas M., Palermo M.S., Protection of Mice Against Shiga Toxin 2 (Stx2)-Associated Damage by Maternal Immunization with a Brucella Lumazine Synthase-Stx2 B Subunit Chimera, Infect Immun, 2(4): 1491-1499 (2014).
[10] Klinke S., Zylberman V., Bonomi H.R., Haase I., Guimaraes B.G., Braden B.C, Bacher A., Fischer M., Goldbaum F.A., Structural and Kinetic Properties of Lumazine Synthase Isoenzymes in the Order Rhizobiales, J Mol Biol., 373(3):664-680 (2007).
[11] Zhang X., Konarev P.V., Petoukhov M.V., Svergun D.I., Xing L., Cheng R.H., Haase I., Fischer M., Bacher A., Ladenstein R., Meining W., Multiple Assembly States of Lumazine Synthase: A Model Relating Catalytic Function and Molecular Assembly, J. Mol. Biol., 362(4): 753-770 (2006).
[12] Klinke S., Zylberman V., Vega D.R., Guimaraes B.G., Braden B.C., Goldbaum F.A., Crystallographic Studies on Decameric Brucella spp. Lumazine Synthase: A Novel Quaternary Arrangement Evolved for a New Function, J. Mol. Biol., 353(1): 124-137 (2005).
[13] Zylberman V., Craig P.O., Klinke S., Braden B.C., Cauerhff A., Goldbaum F.A., High Order Quaternary Arrangement Confers Increased Structural Stability to Brucella sp. Lumazine Synthase, J. Biol. Chem., 279(9): 8093-8101 (2004).
[14] Fornasari M.S., Laplagne D.A., Frankel N., Cauerhff A.A., Goldbaum F.A, Echave J., Sequence Determinants of Quaternary Structure in Lumazine Synthase, Mol Biol Evol., 21(1): 97-107 (2004).
[15] Laplagne D.A., Zylberman V., Ainciart N., Steward M.W., Sciutto E., Fossati C.A., Goldbaum F.A., Engineering of a Polymeric Bacterial Protein as a Scaffold for the Multiple Display of Peptides. Proteins: Structure, Function, and Bioinformatics, 57(4):820-828 (2004).
[17] Azuma Y., Hilvert D., Enzyme Encapsulation in an Engineered Lumazine Synthase Protein Cage, Methods Mol Biol., 1798: 39-55 (2018).
[18] Mejias M.P., Ghersi G., Craig P.O., Panek C.A., Bentancor L.V., Baschkier A., Goldbaum P.F.A., Zylberman V., Palermo M.S., Immunization with a Chimera Consisting of the B Subunit of Shiga Toxin Type 2 and Brucella Lumazine Synthase Confers Total Protection Against Shiga Toxins in Mice, J. Immunol., 191(5): 2403-2411 (2013).
[19] Goldbaum F.A., Velikovsky C.A., Baldi P.C., Mortl S., Bacher A., Fossati C.A., The 18-kDa Cytoplasmic Protein of Brucella Species --An Antigen Useful For Diagnosis--Is a Lumazine Synthase, J Med Microbiol., 48(9): 833-839 (1999).
[20] Berguer P.M., Alzogaray V.A., Rossi A.H., Mundiñano J., Piazzon I., Goldbaum F.A., A Polymeric Protein Induces Specific Cytotoxicity in a TLR4 Dependent Manner in the Absence of Adjuvants, PloS one., 7(9): e45705 (2012).
[21] Akbari R., Sekhavati M.H., Bahrami A., Majidzadeh Heravi R., Yousefi S., Production of Brucella lumazine Synthase Recombinant Protein to Design a Subunit Vaccine against Undulant Fever. Arch Razi Inst., 74(1): 1-6 (2019).
[22] Wei Y., Kumar P., Wahome N., Mantis N.J., Middaugh C.R., Biomedical Applications of Lumazine Synthase, J. Pharm. Sci., 107(9): 2283-2296 (2018).
[23] Rossi A.H., Farias A., Fernandez J.E., Bonomi H.R., Goldbaum F.A., Berguer P.M., Brucella spp. Lumazine Synthase Induces a TLR4-Mediated Protective Response against B16 Melanoma in Mice, PLoS One., 10(5): e0126827 (2015).
[25] Alfano E.F., Lentz E.M., Bellido D., Dus Santos M.J., Goldbaum F.A., Wigdorovitz A., Bravo-Almonacid F.F., Expression of the Multimeric and Highly Immunogenic Brucella spp. Lumazine Synthase Fused to Bovine Rotavirus VP8d as a Scaffold
for Antigen Production in Tobacco Chloroplasts
, Front Plant Sci., 6:1170 (2015).