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New recombinant producer of human ω-amidase based on Escherichia coli

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Abstract

A new artificial gene encoding human ω-amidase (Nit2) adapted for highly efficient expression in E. coli has been established. A pQE-Nit2 plasmid construct controlled by the T5 promoter has been engineered for its expression. The nit2 gene within the pQE-Nit2 construct has optimized codon usage and an artificial 6His-tag sequence inserted directly after the ATG initiation codon. This tag provides the possibility of single-step purification of a product via metal chelate chromatography. The codon-usage optimization involves the inclusion of several codons of extremely rare occurrence in natural E. coli ORFs within a 30 a.a-long N-terminal region. Other codons included in the N-terminus have moderate occurrence in E. coli. The subsequent sequence of the artificial gene has been composed of the most frequently occurring codons in E. coli. The recombinant producer based on the pQE-Nit2 construct allowed purification of the enzyme with an activity of 6.2 ± 0.2 μmol/min/mg protein, which corresponds to or slightly exceeds the specific activity of rat liver Nit2. The omega-amidase preparation is necessary for the screening of potential inhibitors that can be used as candidate drugs to cure hyperammonemia disorders in liver pathologies and oncological diseases.

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References

  1. Bork, P. and Koonin, E.V., Protein Sci., 1994, vol. 3, no. 8, pp. 1344–1346.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Krasnikov, B.F., Chien, C.H., Nostramo, R., Pinto, J.T., Nieves, E., Callaway, M., Sun, J., Huebner, K., and Cooper, A.J., Biochimie, 2009, vol. 91, no. 9, pp. 1072–1080.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Pace, H.C. and Brenner, C., Genome Biol., 2001, vol. 2, no. 1, REVIEWS0001.

    Google Scholar 

  4. Brenner, C., Curr. Opin. Struct. Biol., 2002, vol. 12, no. 6, pp. 775–782.

    Article  CAS  PubMed  Google Scholar 

  5. Cooper, A.J., Shurubor, Y.I., Dorai, T., Pinto, J.T., Isakova, E.P., Deryabina, Y.I., Denton, T.T., and Krasnikov, B.F., Amino Acids, 2016, vol. 48, no. 1, pp. 1–20.

    Article  CAS  PubMed  Google Scholar 

  6. Vergara, F., Duffy, T.E., and Plum, F., Trans. Assoc. Am. Phys., 1973, vol. 86, pp. 255–263.

    CAS  PubMed  Google Scholar 

  7. Vergara, F., Plum, F., and Duffy, T.E., Science, 1974, vol. 183, no. 4120, pp. 81–83.

    Article  CAS  PubMed  Google Scholar 

  8. Hindfelt, B., Plum, F., and Duffy, T.E., J. Clin. Invest., 1977, vol. 59, no. 3, pp. 386–396.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Pasantes-Morales, H. and Franco, R., Astrocyte cellular swelling: mechanisms and relevance to brain edema, in The Role of Glia in Neurotoxicity, 2nd ed., Aschner, M. and Costa, L.G., Eds., Boca Raton, Florida: CRC Press, 2004, pp. 173–190.

  10. Gabuzda, G.J., Phillips, G.B., and Davidson, C.S., New Eng. J. Med., 1952, vol. 246, no. 4, pp. 124–130.

    Article  PubMed  Google Scholar 

  11. Phillips, G.B., Schwartz, R., Gabuzda, G.J., and Davidson, C.S., New Eng. J. Med., 1952, vol. 247, no. 7, pp. 239–246.

    Article  CAS  PubMed  Google Scholar 

  12. Msall, M., Batshaw, M.L., Suss, R., Brusilow, S.W., and Mellits, E.D., New Eng. J. Med., 1984, vol. 310, no. 23, pp. 1500–1505.

    Article  CAS  PubMed  Google Scholar 

  13. Structural insights into yeast Nit2: C169s mutant of yeast Nit2 in complex with an endogenous peptide-like ligand (hydrolase, EC: 3.5.-.-) taxonomy: Saccharomyces cerevisiae S288c proteins: 2 chemicals: 13 modified: 2014/01/25 NCBI Resources.

  14. Liu, H., Gao, Y., Zhang, M., Qiu, X., Cooper, A.J., Niu, L., and Teng, M., Acta Cryst. Sec. D, Biol. Cryst., 2013, vol. 69, pp. 1470–1481.

    Article  CAS  Google Scholar 

  15. Semba, S., Han, S.Y., Qin, H.R., McCorkell, K.A., Iliopoulos, D., Pekarsky, Y., Druck, T., Trapasso, F., Croce, C.M., and Huebner, K., J. Biol. Chem., 2006, vol. 281, no. 38, pp. 28244–28253.

    Article  CAS  PubMed  Google Scholar 

  16. Lin, C.H., Chung, M.Y., Chen, W.B., and Chien, C.H., FEBS J., 2007, vol. 274, no. 11, pp. 2946–2956.

    Article  CAS  PubMed  Google Scholar 

  17. Jay, E., MacKnight, D., Lutze-Wallace, C., Harrison, D., Wishart, P., Liu, W.Y., Asundi, V., Pomeroy-Cloney, L., Rommens, J., et al., J. Biol. Chem., 1984, vol. 259, no. 10, pp. 6311–6317.

    CAS  PubMed  Google Scholar 

  18. Zheng, B., Chai, R., and Yu, X.Int., J. Mol. Med., 2015, vol. 35, no. 5, pp. 1317–1322.

    CAS  Google Scholar 

  19. Mandel, M. and Higa, A., J. Mol. Biol., 1970, vol. 53, no. 1, pp. 159–162.

    Article  CAS  PubMed  Google Scholar 

  20. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J., J. Biol. Chem., 1951, vol. 193, no. 1, pp. 265–275.

    CAS  PubMed  Google Scholar 

  21. Davis, J.B., Ann. N.Y. Acad. Sci., 1964, vol. 121, pp. 404–427.

    Article  CAS  PubMed  Google Scholar 

  22. Krasnikov, B.F., Nostramo, R., Pinto, J.T., and Cooper, A.J., Anal. Biochem., 2009, vol. 391, no. 2, pp. 144–150.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Cooper, A.J., J. Biol. Chem., 1977, vol. 252, no. 6, pp. 2032–2038.

    CAS  PubMed  Google Scholar 

  24. Chung, B.K. and Lee, D.Y., BMC Syst. Biol., 2012, vol. 6, p. 134.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Jaisson, S., Veiga-da-Cunha, M., and van Schaftingen, E., Biochimie, 2009, vol. 91, no. 9, pp. 1066–1071.

    Article  CAS  PubMed  Google Scholar 

  26. Chien, C.H., Gao, Q.Z., Cooper, A.J., Lyu, J.H., and Sheu, S.Y., J. Biol. Chem., 2012, vol. 287, no. 31, pp. 25715–25726.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, 119071, Russia

    B. F. Krasnikov, Yu. I. Deryabina, E. P. Isakova & A. N. Antipov

  2. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, 119334, Russia

    Iu. K. Biriukova & A. B. Shevelev

Authors
  1. B. F. Krasnikov
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  2. Yu. I. Deryabina
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  3. E. P. Isakova
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  4. Iu. K. Biriukova
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  5. A. B. Shevelev
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  6. A. N. Antipov
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Corresponding author

Correspondence to Yu. I. Deryabina.

Additional information

Original Russian Text © B.F. Krasnikov, Yu.I. Deryabina, E.P. Isakova, Iu.K. Biriukova, A.B. Shevelev, A.N. Antipov, 2017, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2017, Vol. 53, No. 3, pp. 271–277.

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Krasnikov, B.F., Deryabina, Y.I., Isakova, E.P. et al. New recombinant producer of human ω-amidase based on Escherichia coli . Appl Biochem Microbiol 53, 290–295 (2017). https://doi.org/10.1134/S0003683817030115

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  • DOI: https://doi.org/10.1134/S0003683817030115

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