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Analysis of Activity of Human Steroidogenic Acute Regulatory Protein (STARD1) Expressed in Escherichia coli Cells

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Abstract

One of the main obstacles to the successful use of Escherichia coli cells for steroid transformation in biotechnological processes is inefficient transport of steroid substrates into the cells. Here, we tested the possibility of using human cholesterol transfer protein STARD1 (steroidogenic acute regulatory protein) to increase the efficiency of steroid uptake by bacterial cells. Genetic constructs were obtained for the synthesis in E. coli BL21 (DE3) cells of a truncated version of STARD1 containing protein functional domain (residues 66-285) and STARD1 (66-285)-GFP fusion protein, both carrying bacterial periplasmic targeting sequence pelB at the N-terminus. Analysis of preparations of E. coli/pET22b/STARD1-GFP cells by fluorimetry and Western blotting confirmed that the used expression system ensured the synthesis of the heterologous protein. Using fluorescence spectroscopy, it was demonstrated that the presence of STARD1 in the cells increased the efficiency of assimilation of NBD-labeled cholesterol analogues by E. coli/pET22b/STARD1 cells 1.3-1.6 times (p < 0.05) compared to the wild-type cells, thus demonstrating that human STARD1 exhibits its functional activity in bacterial cells. This opens prospects for optimizing and using a fundamentally new approach to increase the efficiency of steroid uptake by cells – the inclusion of a specific carrier protein in the cell membrane, which can expand the arsenal of methods used to obtain strains of microorganisms for synthesis.

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Abbreviations

20NP:

20-(NBD)amino-pregn-5-en-3β-ol

GFP:

green fluorescent protein

MCD:

methyl-β-cyclodextrin

NBD:

nitrobenzoxadiazole

pelB:

N-terminal signaling sequence of pectate lyase B from Erwinia carotovora

STARD1:

steroidogenic acute regulatory protein

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Acknowledgments

This work was supported by the Russian Foundation for Basic Research (project no. 20-08-00467-a). The authors are grateful to N. N. Sluchanko for kindly providing pET-22b(+) and H-MBP-3C-STARD166-285 plasmids.

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Authors and Affiliations

  1. Faculty of Biology, Lomonosov Moscow State University, 119234, Moscow, Russia

    Sofia V. Zamalutdinova, Aleksei V. Zamalutdinov & Mikhail A. Rubtsov

  2. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119234, Moscow, Russia

    Ludmila V. Isaeva & Ludmila A. Novikova

  3. Research Institute for Physical Chemical Problems, Belarusian State University, 220030, Minsk, Belarus

    Yaroslav V. Faletrov

  4. Sechenov First Moscow State Medical University, 119991, Moscow, Russia

    Mikhail A. Rubtsov

Authors
  1. Sofia V. Zamalutdinova
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  2. Ludmila V. Isaeva
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  3. Aleksei V. Zamalutdinov
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  4. Yaroslav V. Faletrov
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  5. Mikhail A. Rubtsov
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  6. Ludmila A. Novikova
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Contributions

L. A. Novikova and M. A. Rubtsov developed the concept, supervised the study, and wrote the manuscript; S. V. Zamalutdinova, L. V. Isaeva, A. V. Zamalutdinov, Y. V. Faletrov, and L. A. Novikova conducted the experiments; A. V. Zamalutdinov performed statistical analysis of results.

Corresponding authors

Correspondence to Sofia V. Zamalutdinova or Ludmila A. Novikova.

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The authors declare no conflicts of interest. This article contains no description of studies using human or animal subjects performed by any of the authors.

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Zamalutdinova, S.V., Isaeva, L.V., Zamalutdinov, A.V. et al. Analysis of Activity of Human Steroidogenic Acute Regulatory Protein (STARD1) Expressed in Escherichia coli Cells. Biochemistry Moscow 87, 1015–1020 (2022). https://doi.org/10.1134/S0006297922090127

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  • DOI: https://doi.org/10.1134/S0006297922090127

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