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ORF19.2286 Gene: Isolation and Purification of Deoxyhypusine Hydroxylase from the Human Pathogenic Yeast Candida albicans

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Abstract

Candida albicans (C. albicans) is a fungal pathogen that causes infections of the wet body surfaces and the blood in immunocompromised patients or individuals with imbalanced microflora. Since the cases of clinically meaningful candidosis are on the rise, efficient C. albicans therapy is in a high demand. Informed drug design requires well-characterized C. albicans targets, including these aimed at disrupting its post-translational modifications. C. albicans ORF19.2286 gene encodes an ortholog of human deoxyhypusine hydroxylase (DOHH). Here, this ORF was cloned from the SC5314 strain and re-expressed in Escherichia coli as sGB1-CaDOHH construct with 6×His tag on the N-terminus of the fusion protein, then purified, and GB1-tag was removed with Tobacco etch virus (TEV) protease. Several amino acid sequence differences between C. albicans and animal DOHHs were noted, and are useful for a selection of the binding sites for antimicrobials in CaDOHH. We present the protocol for the heterologous expression and purification of C. albicans DOHH, which is suitable for further crystallization.

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Funding

This work was supported by the Russian Science Foundation grant no. 20-65-47031.

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Authors and Affiliations

  1. Kazan Federal University, 420008, Kazan, Russia

    E. Agboigba, E. Kuchaev, N. Garaeva, E. Klochkova, A. Varfolomeev, K. Usachev, M. Yusupov & Sh. Validov

  2. Kazan Scientific Center, Russian Academy of Sciences, 420008, Kazan, Russia

    E. Kuchaev, K. Usachev & Sh. Validov

  3. Institute of Genetics and Molecular and Cellular Biology (IGBMC), 67400, Illkirch-Graffenstaden, France

    M. Yusupov

Authors
  1. E. Agboigba
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  2. E. Kuchaev
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  3. N. Garaeva
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  4. E. Klochkova
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  5. A. Varfolomeev
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  6. K. Usachev
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  7. M. Yusupov
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  8. Sh. Validov
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Correspondence to K. Usachev or Sh. Validov.

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Conflict of interests. The authors declare that they have no conflicts of interest.

Statement on the welfare of humans or animals. This article does not contain any studies involving humans or animals performed by any of the authors.

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The text was submitted by the author(s) in English.

Abbreviations: CRB, Cell Resuspension Buffer; GB1, B1 domain of streptococcal protein G; DHS, deoxyhypusine synthase; DOHH, deoxyhypusine hydroxylase; TEV, Tobacco etch virus; PAGE, polyacrylamide gel electrophoresis; aa, amino acids.

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Agboigba, E., Kuchaev, E., Garaeva, N. et al. ORF19.2286 Gene: Isolation and Purification of Deoxyhypusine Hydroxylase from the Human Pathogenic Yeast Candida albicans. Mol Biol 56, 269–275 (2022). https://doi.org/10.1134/S0026893322020029

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