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Archives of Virology

, Volume 154, Issue 2, pp 337–341 | Cite as

Restoration of potato virus X coat protein capacity for assembly with RNA after His-tag removal

  • Olga Zayakina
  • Marina Arkhipenko
  • Alexander Smirnov
  • Nina Rodionova
  • Olga Karpova
  • Joseph Atabekov
Brief Report

Abstract

We found that the fusion of hexahistidine (H)6 tag to the potato virus X (PVX) coat protein (CP) abolished its ability to be assembled with viral RNA into helical nucleoprotein virus-like particles (VLPs). Instead, irregular agglomerates were produced upon incubation of PVX RNA with (H)6-tagged PVX CP. A factor Xa recognition site, IEGR, was inserted upstream of the CP coding sequence. Removal of the (H)6 tag from PVX CP by Xa protease restored its ability to bind RNA and to assemble VLPs. In addition to the canonical IEGR site, the factor Xa protease was found to cleave PVX CP at a second (non-consensus) site, AVTRGR, located close to the C-terminus of PVX CP. The latter cleavage did not affect reassembly of the PVX RNA and CP into VLPs.

Keywords

Coat Protein Recombinant Coat Protein Coat Protein Assembly Additional Cleavage Site Coat Protein Code Sequence 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We are grateful to Dr. I. B. Kaplan for reading the manuscript and editorial comments. This work was funded in part by the Federal Agency of Science and Innovation (FASI) (contract No 02.512.11.2103), and the Russian Foundation for Basic Research (Grant 06-04-48-176),

References

  1. 1.
    Atabekov JG, Rodionova NP, Karpova OV, Kozlovsky SV, Novikov VK, Arkhipenko MV (2001) Translational activation of encapsidated potato virus X RNA by coat protein phosphorylation. Virology 286:466–474PubMedCrossRefGoogle Scholar
  2. 2.
    Blanc S, Ammar ED, Garcia-Lampasona S, Dolja VV, Llave C, Baker J, Pirone TP (1998) Mutations in the potyvirus helper component protein: effects on interactions with virions and aphid stylets. J Gen Virol 79:3119–3122PubMedGoogle Scholar
  3. 3.
    Chapman SN, Hills G, Watts J, Baulcombe DC (1992) Mutational analysis of the coat protein gene of potato virus X. Effect of virion morphology and viral pathogenicity. Virology 191:223–230PubMedCrossRefGoogle Scholar
  4. 4.
    Goel A, Colcher D, Koo JS, Booth BJ, Pavlinkova G, Batra SK (2000) Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct. Biochim Biophys Acta 1523(1):13–20PubMedGoogle Scholar
  5. 5.
    Goodman RM (1975) Reconstitution of potato virus X in vitro. I. Properties of the dissociated protein structural subunits. Virology 68(2):287–298PubMedCrossRefGoogle Scholar
  6. 6.
    Jenny RJ, Mann KG, Lundblad RL (2003) A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa. Protein Expr Purif 31(1):1–11PubMedCrossRefGoogle Scholar
  7. 7.
    Hung M, Gibbs CS, Tsiang M (2002) Biochemical characterization of rhinovirus RNA-dependent RNA polymerase. Antiviral Res 56:99–114PubMedCrossRefGoogle Scholar
  8. 8.
    Karpova OV, Ivanov KI, Rodionova NP, Dorokhov YuL, Atabekov JG (1997) Nontranslatability and dissimilar behavior in plants and protoplasts of viral RNA and movement protein complexes formed in vitro. Virology 230:11–21PubMedCrossRefGoogle Scholar
  9. 9.
    Karpova OV, Zayakina OV, Arkhipenko MV, Sheval EV, Kiselyova OI, Poljakov VYu, Yaminsky IV, Rodionova NP, Atabekov JG (2006a) Potato virus X RNA-mediated assembly of single-tailed ternary complex “coat protein–RNA–movement protein”. J Gen Virol 87:2731–2740PubMedCrossRefGoogle Scholar
  10. 10.
    Karpova OV, Arkhipenko MV, Zayakina OV, Nikitin NA, Kiselyova OI, Kozlovsky SV, Rodionova NP, Atabekov JG (2006b) Regulation of RNA translation in Potato virus X RNA–coat protein complexes: the key role of the N-terminal segment of the protein. Mol Biol (Russia) 4:628–634CrossRefGoogle Scholar
  11. 11.
    Kenig M, Peternel S, Gaberc-Porekar V, Menart V (2006) Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins. J Chromatogr A 1101(1–2):293–306PubMedCrossRefGoogle Scholar
  12. 12.
    Kowolik CM, Hengstenberg W (1998) The lactose transporter of Staphylococcus aureus-overexpression, purification and characterization of the histidine-tagged domains IIC and IIB. Eur J Biochem 228:798–804Google Scholar
  13. 13.
    Kwon SJ, Park MR, Kim KW, Plante CA, Hemenway CL, Kim KH (2005) cis-Acting sequences required for coat protein binding and in vitro assembly of Potato virus X. Virology 334(1):83–97PubMedCrossRefGoogle Scholar
  14. 14.
    Mohanty AK, Wiener MC (2004) Membrane protein expression and production: effects of polyhistidine tag length and position. Protein Expr Purif 33:311–325PubMedCrossRefGoogle Scholar
  15. 15.
    Rodionova NP, Karpova OV, Kozlovsky SV, Zayakina OV, Arkhipenko MV, Atabekov JG (2003) Linear remodeling of helical virus by movement protein binding. J Mol Biol 333:565–572PubMedCrossRefGoogle Scholar
  16. 16.
    Routzahn KM, Waugh DS (2002) Differential effects of supplementary affinity tags on the solubility of MBP fusion proteins. J Struct Funct Genomics 2(2):83–92PubMedCrossRefGoogle Scholar
  17. 17.
    Votchitseva YuA, Efremenko EN, Aliev TK, Varfolomeyev SD (2006) Properties of hexahistidine-tagged organophosphate hydrolase. Biochemistry (Moscow) 71(N2):167–172CrossRefGoogle Scholar
  18. 18.
    Woestenenk EA, Hammarström M, van den Berg S, Härd T, Berglund H (2004) His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors. J Struct Funct Genomics 5(3):217–229PubMedCrossRefGoogle Scholar
  19. 19.
    Yang SJ, Park NH, Lee TH, Cha J (2002) Expression, purification and characterization of a recombinant levan fructotransferase. Biotechnol Appl Biochem 35(3):199–203PubMedCrossRefGoogle Scholar
  20. 20.
    Zayakina OV, Arkhipenko MV, Kozlovsky SV, Nikitin NA, Smirnov AV, Susi P, Rodionova NP, Karpova OV, Atabekov JG (2008) Mutagenic analysis of Potato Virus X movement protein (TGBp1) and the coat protein (CP): in vitro TGBp1-CP binding and viral RNA translation activation. Mol Plant Pathol 9(1):37–44PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Olga Zayakina
    • 1
  • Marina Arkhipenko
    • 1
  • Alexander Smirnov
    • 1
  • Nina Rodionova
    • 1
  • Olga Karpova
    • 1
  • Joseph Atabekov
    • 1
    • 2
  1. 1.Department of VirologyMoscow State UniversityMoscowRussia
  2. 2.A. N. Belozersky Institute of Physico-Chemical BiologyMoscow State UniversityMoscowRussia

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