In vivo method for biotinylation of recombinant variola virus proteins

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Abstract

The work implements a method for specific in vivo biotinylation of recombinant proteins M1 and B7 of the variola virus during biosynthesis in CHO-K1 cells. To do this, co-expression of the biotin ligase BirA and target genes encoding the ectodomains of the M1 and B7 proteins with a C-terminal avi-tag was carried out in CHO-K1 cells in the presence of biotin in the culture medium. The optimal biotin concentration for the expression of M1 and B7 proteins was 125 μM. The production of biotinylated recombinant proteins has been complicated by low yields. To increase the production of target proteins, low molecular weight enhancers were added to the culture medium: lithium acetate, sodium valproate and caffeine. The enhancers increased the yield of the target protein by 1.3–4.9 times and did not affect the efficiency of biotinylation. The highest yield of biotinylated protein was achieved with the simultaneous addition of a concentration of 10 mM lithium acetate and 2.5 mM sodium valproate.

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About the authors

V. N. Nikitin

State Research Center of Virology and Biotechnology Vector

Email: dnshcherbakov@gmail.com
Russian Federation, Koltsovo, 630559

Yu. A. Merkuleva

State Research Center of Virology and Biotechnology Vector

Email: dnshcherbakov@gmail.com
Russian Federation, Koltsovo, 630559

D. N. Shcherbakov

State Research Center of Virology and Biotechnology Vector; Altai State University

Author for correspondence.
Email: dnshcherbakov@gmail.com
Russian Federation, Koltsovo, 630559; Barnaul, 656049

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Supplementary files

Supplementary Files
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2. Fig. 1. Map of the plasmid vector pVEAL-BirA (a) and the sequence of the BirA gene expression cassette (b).

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3. Fig. 2. Map of the plasmid vector pVEAL2 (a) and the M1R (b) and B7R (c) genes containing the secretion signal peptide, his-tag and avi-tag tags.

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4. Fig. 3. Viability of CHO-K1 (1) and CHO-BirA (2) cells when cultured in the presence of different concentrations of biotin.

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5. Fig. 4. Western blot analysis of biotinylated proteins: M1 (a) and B7 (b), 1, 3 – isolated from the culture medium with the addition of exogenous biotin, 2, 4 – from the culture medium without biotin, M – marker.

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6. Fig. 5. Biotinylation level (%) of the B7 protein of variola virus depending on the biotin concentration in the culture medium. The values ​​are normalized to the maximum optical density.

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7. Fig. 6. The amount of total and biotinylated recombinant protein B7 (a) and M1 (b) of smallpox virus in the culture medium, depending on the concentration of enhancers in the medium.

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