A Generic System for the Expression and Purification of Soluble and Stable Influenza Neuraminidase

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2011 Feb 18

PMID 21326879

Citations 29

Authors

Peter M Schmidt

Rebecca M Attwood

Peter G Mohr

Susan A Barrett

Jennifer L McKimm-Breschkin

Affiliations

Soon will be listed here.

Abstract

The influenza surface glycoprotein neuraminidase (NA) is essential for the efficient spread of the virus. Antiviral drugs such as Tamiflu (oseltamivir) and Relenza (zanamivir) that inhibit NA enzyme activity have been shown to be effective in the treatment of influenza infections. The recent 'swine flu' pandemic and world-wide emergence of Tamiflu-resistant seasonal human influenza A(H1N1) H(274)Y have highlighted the need for the ongoing development of new anti-virals, efficient production of vaccine proteins and novel diagnostic tools. Each of these goals could benefit from the production of large quantities of highly pure and stable NA. This publication describes a generic expression system for NAs in a baculovirus Expression Vector System (BEVS) that is capable of expressing milligram amounts of recombinant NA. To construct NAs with increased stability, the natural influenza NA stalk was replaced by two different artificial tetramerization domains that drive the formation of catalytically active NA homotetramers: GCN4-pLI from yeast or the Tetrabrachion tetramerization domain from Staphylothermus marinus. Both recombinant NAs are secreted as FLAG-tagged proteins to allow for rapid and simple purification. The Tetrabrachion-based NA showed good solubility, increased stability and biochemical properties closer to the original viral NA than the GCN4-pLI based construct. The expressed quantities and high quality of the purified recombinant NA suggest that this expression system is capable of producing recombinant NA for a broad range of applications including high-throughput drug screening, protein crystallisation, or vaccine development.

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References

Harbury P, Zhang T, Kim P, Alber T . A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science. 1993; 262(5138):1401-7. DOI: 10.1126/science.8248779. View

Becker E, Schmidt P, Schramm M, Schroder H, Walter U, Hoenicka M . The vasodilator-stimulated phosphoprotein (VASP): target of YC-1 and nitric oxide effects in human and rat platelets. J Cardiovasc Pharmacol. 2000; 35(3):390-7. DOI: 10.1097/00005344-200003000-00007. View

Yongkiettrakul S, Boonyapakron K, Jongkaewwattana A, Wanitchang A, Leartsakulpanich U, Chitnumsub P . Avian influenza A/H5N1 neuraminidase expressed in yeast with a functional head domain. J Virol Methods. 2008; 156(1-2):44-51. PMC: 7112848. DOI: 10.1016/j.jviromet.2008.10.025. View

Johnson G, Jiang W . Characterization of cathepsin L secreted by Sf21 insect cells. Arch Biochem Biophys. 2005; 444(1):7-14. DOI: 10.1016/j.abb.2005.09.011. View

Mittl P, Deillon C, Sargent D, Liu N, Klauser S, Thomas R . The retro-GCN4 leucine zipper sequence forms a stable three-dimensional structure. Proc Natl Acad Sci U S A. 2000; 97(6):2562-6. PMC: 15968. DOI: 10.1073/pnas.97.6.2562. View

Lawrence M, Borg N, Streltsov V, Pilling P, Epa V, Varghese J . Structure of the haemagglutinin-neuraminidase from human parainfluenza virus type III. J Mol Biol. 2004; 335(5):1343-57. DOI: 10.1016/j.jmb.2003.11.032. View

Blick T, Tiong T, Sahasrabudhe A, Varghese J, Colman P, Hart G . Generation and characterization of an influenza virus neuraminidase variant with decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac2en. Virology. 1995; 214(2):475-84. DOI: 10.1006/viro.1995.0058. View

Yano T, Nobusawa E, Nagy A, Nakajima S, Nakajima K . Effects of single-point amino acid substitutions on the structure and function neuraminidase proteins in influenza A virus. Microbiol Immunol. 2008; 52(4):216-23. DOI: 10.1111/j.1348-0421.2008.00034.x. View

McKimm-Breschkin J, Sahasrabudhe A, Blick T, McDonald M, Colman P, Hart G . Mutations in a conserved residue in the influenza virus neuraminidase active site decreases sensitivity to Neu5Ac2en-derived inhibitors. J Virol. 1998; 72(3):2456-62. PMC: 109546. DOI: 10.1128/JVI.72.3.2456-2462.1998. View

10.

Stetefeld J, Jenny M, Schulthess T, Landwehr R, Engel J, Kammerer R . Crystal structure of a naturally occurring parallel right-handed coiled coil tetramer. Nat Struct Biol. 2000; 7(9):772-6. DOI: 10.1038/79006. View

11.

Collins P, Haire L, Lin Y, Liu J, Russell R, Walker P . Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants. Nature. 2008; 453(7199):1258-61. DOI: 10.1038/nature06956. View

12.

Castrucci M, Kawaoka Y . Biologic importance of neuraminidase stalk length in influenza A virus. J Virol. 1993; 67(2):759-64. PMC: 237428. DOI: 10.1128/JVI.67.2.759-764.1993. View

13.

Takahashi T, Suzuki Y, Nishinaka D, KAWASE N, Kobayashi Y, Hidari K . Duck and human pandemic influenza A viruses retain sialidase activity under low pH conditions. J Biochem. 2001; 130(2):279-83. DOI: 10.1093/oxfordjournals.jbchem.a002983. View

14.

Monto A, McKimm-Breschkin J, Macken C, Hampson A, Hay A, Klimov A . Detection of influenza viruses resistant to neuraminidase inhibitors in global surveillance during the first 3 years of their use. Antimicrob Agents Chemother. 2006; 50(7):2395-402. PMC: 1489772. DOI: 10.1128/AAC.01339-05. View

15.

Kuhnel K, Jarchau T, Wolf E, Schlichting I, Walter U, Wittinghofer A . The VASP tetramerization domain is a right-handed coiled coil based on a 15-residue repeat. Proc Natl Acad Sci U S A. 2004; 101(49):17027-32. PMC: 535362. DOI: 10.1073/pnas.0403069101. View

16.

Oakley A, Barrett S, Peat T, Newman J, Streltsov V, Waddington L . Structural and functional basis of resistance to neuraminidase inhibitors of influenza B viruses. J Med Chem. 2010; 53(17):6421-31. PMC: 2932999. DOI: 10.1021/jm100621s. View

17.

Fiers W, Neirynck S, Deroo T, Saelens X, Jou W . Soluble recombinant influenza vaccines. Philos Trans R Soc Lond B Biol Sci. 2002; 356(1416):1961-3. PMC: 1088575. DOI: 10.1098/rstb.2001.0980. View

18.

Fernley R, Wright R, Coghlan J . Complete amino acid sequence of ovine salivary carbonic anhydrase. Biochemistry. 1988; 27(8):2815-20. DOI: 10.1021/bi00408a023. View

19.

Aeed P, Elhammer A . Glycosylation of recombinant prorenin in insect cells: the insect cell line Sf9 does not express the mannose 6-phosphate recognition signal. Biochemistry. 1994; 33(29):8793-7. DOI: 10.1021/bi00195a022. View

20.

Deroo T, Jou W, Fiers W . Recombinant neuraminidase vaccine protects against lethal influenza. Vaccine. 1996; 14(6):561-9. DOI: 10.1016/0264-410x(95)00157-v. View