Efficient soluble expression and purification of influenza A and B nucleoproteins in E. coli

Полный текст:


Viral nucleoprotein (NP) is an abundant essential protein of an influenza virus that has important functional and structural roles. It participates in genomic organization, nuclear trafficking, RNA transcription, and genome replication. From the research point of view, NP is an important protein that is used in the development of new diagnostic methods and vaccination protocols. NP is a promising target for antiviral chemotherapeutic drugs as well. Successful expression of codon-optimized NP genes in E. coli has been reported. In this study, we demonstrated the efficient expression and purification of soluble NPs of influenza A and B viruses in E. coli without the codon-optimization of DNA sequences. This procedure preserves the co-translational protein folding, protein configuration and function. Obtained NPs of influenza A and B viruses were monomers and reacted well with mouse specific antibodies according to Western blot analysis. Our results show that both influenza A and influenza B virus NPs can be efficiently expressed in E. coli without codon-optimization.

Об авторах

N. D. Yolshin
Smorodintsev Research Institute of Influenza

Saint Petersburg

A. A. Shaldzhyan
Smorodintsev Research Institute of Influenza

Saint Petersburg

S. A. Klotchenko
Smorodintsev Research Institute of Influenza

Saint Petersburg

Список литературы

1. Gorman OT, Bean WJ, Kawaoka Y, Webster RG. Evolution of the nucleoprotein gene of influenza A virus. J Virol. 1990; 64(4), 1487-97. PubMed PMID: 2319644.

2. Shu LL, Bean WJ, Webster RG. Analysis of the evolution and variation of the human influenza A virus nucleoprotein gene from 1933 to 1990. J Virol. 1993; 67(5), 2723-9. PubMed PMID: 8474171.

3. O’Neill RE, Jaskunas R, Blobel G, Palese P, Moroianu J. Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein and transport factors required for protein import. J Biol Chem. 1995; 270(39), 22701-4. doi: 10.1074/jbc.270.39.22701.

4. Turrell L, Lyall JW, Tiley LS, Fodor E, Vreede FT. The role and assembly mechanism of nucleoprotein in influenza A virus ribonucleoprotein complexes. Nat Commun. 2013; 4, 1591. doi: 10.1038/ncomms2589.

5. Eisfeld AJ, Neumann G, Kawaoka Y. At the centre: influenza A virus ribonucleoproteins. Nat Rev Microbiol. 2015; 13(1):, 28-41. doi: 10.1038/nrmicro3367.

6. Baudin F, Bach C, Cusack S, Ruigrok RW. Structure of influenza virus RNP. I. Influenza virus nucleoprotein melts secondary structure in panhandle RNA and exposes the bases to the solvent. EMBO J. 1994; 13(13), 3158-65. PubMed PMID: 8039508.

7. Compans RW, Content J, Duesberg PH. Structure of the ribonucleoprotein of influenza virus. J Virol. 1972; 10(4), 795-800. PubMed PMID: 4117350.

8. Ortega J, Martin-Benito J, Zurcher T, Valpuesta JM, Carrascosa JL, Ortin J. Ultrastructural and functional analyses of recombinant influenza virus ribonucleoproteins suggest dimerization of nucleoprotein during virus amplification. J Virol. 2000; 74(1), 156-63. doi: 10.1128/jvi.74.1.156-163.2000.

9. Cianci C, Gerritz SW, Deminie C, Krystal M. Influenza nucleoprotein: promising target for antiviral chemotherapy. Antivir Chem Chemother. 2012; 23(3), 77-91. doi: 10.3851/IMP2235.

10. Heiny AT, Miotto O, Srinivasan KN, Khan AM, Zhang GL, Brusic V, et al. Evolutionarily conserved protein sequences of influenza a viruses, avian and human, as vaccine targets. PLoS One. 2007; 2(11), e1190. doi: 10.1371/journal.pone.0001190.

11. Huang B, Wang W, Li R, Wang X, Jiang T, Qi X, et al. Influenza A virus nucleoprotein derived from Escherichia coli or recombinant vaccinia (Tiantan) virus elicits robust cross-protection in mice. Virol J. 2012; 9, 322. doi: 10.1186/1743-422X-9-322.

12. Epstein SL, Kong WP, Misplon JA, Lo CY, Tumpey TM, Xu L, et al. Protection against multiple influenza A subtypes by vaccination with highly conserved nucleoprotein. Vaccine. 2005; 23(46-47), 5404-10. doi: 10.1016/j.vaccine.2005.04.047.

13. Vemula SV, Zhao J, Liu J, Wang X, Biswas S, Hewlett I. Current Approaches for Diagnosis of Influenza Virus Infections in Humans. Viruses. 2016; 8(4), 96. doi: 10.3390/v8040096.

14. Phuong NH, Kwak C, Heo CK, Cho EW, Yang J, Poo H. Development and Characterization of Monoclonal Antibodies against Nucleoprotein for Diagnosis of Influenza A Virus. J Microbiol Biotechnol. 2018; 28(5), 809-15. doi: 10.4014/jmb.1801.01002.

15. Brule CE, Grayhack EJ. Synonymous Codons: Choose Wisely for Expression. Trends Genet. 2017; 33(4), 283-97. doi: 10.1016/j.tig.2017.02.001.

16. Hanson G, Coller J. Codon optimality, bias and usage in translation and mRNA decay. Nat Rev Mol Cell Biol. 2018; 19(1), 20-30. doi: 10.1038/nrm.2017.91.

17. Mauro VP, Chappell SA. Considerations in the Use of Codon Optimization for Recombinant Protein Expression. Methods Mol Biol. 2018; 1850, 275-88. doi: 10.1007/978-1-4939-8730-6_18.

18. Huang BY, Wang WL, Wang XP, Jiang T, Tan WJ, Ruan L. [Efficient soluble expression and purification of influenza A nucleoprotein in Escherichia coli]. Bing Du Xue Bao. 2011; 27(1), 50-7. PubMed PMID: 21462506.

19. Yoon SJ, Park YJ, Kim HJ, Jang J, Lee SJ, Koo S, et al. Optimized Expression, Purification, and Rapid Detection of Recombinant Influenza Nucleoproteins Expressed in Sf9 Insect Cells. J Microbiol Biotechnol. 2018; 28(10), 1683-90. doi: 10.4014/jmb.1805.05053.

20. Mauro VP, Chappell SA. A critical analysis of codon optimization in human therapeutics. Trends Mol Med. 2014; 20(11), 604-13. doi: 10.1016/j.molmed.2014.09.003.

21. Zhao F, Yu CH, Liu Y. Codon usage regulates protein structure and function by affecting translation elongation speed in Drosophila cells. Nucleic Acids Res. 2017; 45(14), 8484-92. doi: 10.1093/nar/gkx501.

22. Kudla G, Murray AW, Tollervey D, Plotkin JB. Codingsequence determinants of gene expression in Escherichia coli. Science. 2009; 324(5924), 255-8. doi: 10.1126/science.1170160.

23. Zhou M, Guo J, Cha J, Chae M, Chen S, Barral JM, et al. Non-optimal codon usage affects expression, structure and function of clock protein FRQ. Nature. 2013; 495(7439), 111-5. doi: 10.1038/nature11833.

24. Konczal J, Bower J, Gray CH. Re-introducing nonoptimal synonymous codons into codon-optimized constructs enhances soluble recovery of recombinant proteins from Escherichia coli. PLoS One. 2019; 14(4), e0215892. doi: 10.1371/journal.pone.0215892.

25. Arora DJ, Tremblay P, Bourgault R, Boileau S. Concentration and purification of influenza virus from allantoic fluid. Anal Biochem. 1985; 144(1), 189-92. doi: 10.1016/0003-2697(85)90103-4.

26. Sharp PM, Li WH. The codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987; 15(3), 1281-95. doi: 10.1093/nar/15.3.1281.

27. Komar AA, Lesnik T, Reiss C. Synonymous codon substitutions affect ribosome traffic and protein folding during in vitro translation. FEBS Lett. 1999; 462(3), 387-91. doi: 10.1016/s0014-5793(99)01566-5.

28. Drummond DA, Wilke CO. Mistranslation-induced protein misfolding as a dominant constraint on coding-sequence evolution. Cell. 2008; 134(2), 341-52. doi: 10.1016/j.cell.2008.05.042.

29. Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, Calcagno AM, Ambudkar SV, et al. A “silent” polymorphism in the MDR1 gene changes substrate specificity. Science. 2007; 315(5811), 525-8. doi: 10.1126/science.1135308.

Для цитирования:

Yolshin N.D., Shaldzhyan A.A., Klotchenko S.A. Efficient soluble expression and purification of influenza A and B nucleoproteins in E. coli. Microbiology Independent Research Journal (MIR Journal). 2019;6(1):43-48.

For citation:

Yolshin N.D., Shaldzhyan A.A., Klotchenko S.A. Efficient soluble expression and purification of influenza A and B nucleoproteins in E. coli. Microbiology Independent Research Journal (MIR Journal). 2019;6(1):43-48.

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