Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

Luis M. Branco, Alex Matschiner, Joseph N. Fair, Augustine Goba, Darryl B. Sampey, Philip J. Ferro, Kathleen A. Cashman, Randal J. Schoepp, Robert B. Tesh, Daniel G. Bausch, Robert F. Garry, Mary C. Guttieri

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Background. There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2). Results. Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP) fusions in the Rosetta strains of Escherichia coli (E. coli) using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC). Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF) against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA). Conclusion. These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.

Original languageEnglish (US)
Article number74
JournalVirology Journal
Volume5
DOIs
StatePublished - 2008

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Lassa virus
Glycoproteins
Maltose-Binding Proteins
Nucleoproteins
Proteins
Old World Arenaviruses
New World Arenaviruses
Lassa Fever
Amylose
Factor Xa
Ascitic Fluid
Fermentation
Antiviral Agents
Gel Chromatography
Western Blotting
Enzyme-Linked Immunosorbent Assay
Escherichia coli

ASJC Scopus subject areas

  • Virology
  • Genetics(clinical)

Cite this

Branco, L. M., Matschiner, A., Fair, J. N., Goba, A., Sampey, D. B., Ferro, P. J., ... Guttieri, M. C. (2008). Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance. Virology Journal, 5, [74]. https://doi.org/10.1186/1743-422X-5-74

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance. / Branco, Luis M.; Matschiner, Alex; Fair, Joseph N.; Goba, Augustine; Sampey, Darryl B.; Ferro, Philip J.; Cashman, Kathleen A.; Schoepp, Randal J.; Tesh, Robert B.; Bausch, Daniel G.; Garry, Robert F.; Guttieri, Mary C.

In: Virology Journal, Vol. 5, 74, 2008.

Research output: Contribution to journalArticle

Branco, LM, Matschiner, A, Fair, JN, Goba, A, Sampey, DB, Ferro, PJ, Cashman, KA, Schoepp, RJ, Tesh, RB, Bausch, DG, Garry, RF & Guttieri, MC 2008, 'Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance', Virology Journal, vol. 5, 74. https://doi.org/10.1186/1743-422X-5-74
Branco, Luis M. ; Matschiner, Alex ; Fair, Joseph N. ; Goba, Augustine ; Sampey, Darryl B. ; Ferro, Philip J. ; Cashman, Kathleen A. ; Schoepp, Randal J. ; Tesh, Robert B. ; Bausch, Daniel G. ; Garry, Robert F. ; Guttieri, Mary C. / Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance. In: Virology Journal. 2008 ; Vol. 5.
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abstract = "Background. There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2). Results. Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP) fusions in the Rosetta strains of Escherichia coli (E. coli) using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC). Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF) against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA). Conclusion. These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.",
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