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Heterologous expression and isolation of influenza A virus nuclear export protein NEP

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Abstract

Influenza A virus nuclear export protein NEP (NS2, 14.4 kDa) plays a key role in various steps of the virus life cycle. Highly purified protein preparations are required for structural and functional studies. In this study, we designed a series of Escherichia coli plasmid constructs for highly efficient expression of the NEP gene under control of the constitutive trp promoter. An efficient method for extraction of NEP from inclusion bodies based on dodecyl sulfate treatment was developed. Preparations of purified NEP with either N-or C-terminal (His)6-tag were obtained using Ni-NTA agarose affinity chromatography with yield of more than 20 mg per liter of culture. According to CD data, the secondary structure of the proteins matched that of natural NEP. A high propensity of NEP to aggregate over a wide range of conditions was observed.

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Abbreviations

a.a.:

amino acid residue

bp:

base pairs

IPTG:

isopropyl-β-D-thiogalactopyranoside

NEP:

nuclear export protein

NEP-C:

NEP with C-terminal (His)6-tag

NEP-N:

NEP with N-terminal (His)6-tag

PCR:

polymerase chain reaction

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Author information

Authors and Affiliations

  1. Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics, 119991, Moscow, Russia

    A. O. Golovko

  2. Lomonosov Moscow State University, Faculty of Chemistry, 119991, Moscow, Russia

    O. N. Koroleva

  3. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 11991, Moscow, Russia

    V. L. Drutsa

Authors
  1. A. O. Golovko
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  2. O. N. Koroleva
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  3. V. L. Drutsa
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Corresponding author

Correspondence to O. N. Koroleva.

Additional information

Original Russian Text © A. O. Golovko, O. N. Koroleva, V. L. Drutsa, 2017, published in Biokhimiya, 2017, Vol. 82, No. 12, pp. 1907-1916.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM17-334, November 6, 2017.

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Golovko, A.O., Koroleva, O.N. & Drutsa, V.L. Heterologous expression and isolation of influenza A virus nuclear export protein NEP. Biochemistry Moscow 82, 1529–1537 (2017). https://doi.org/10.1134/S0006297917120124

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  • DOI: https://doi.org/10.1134/S0006297917120124

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