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

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Sofia V. Zamalutdinova^1,a*, Ludmila V. Isaeva², Aleksei V. Zamalutdinov¹, Yaroslav V. Faletrov^3,b, Mikhail A. Rubtsov^1,4,c, and Ludmila A. Novikova^2,d*

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

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

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

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

^* To whom correspondence should be addressed.

Received July 17, 2022; Revised August 8, 2022; Accepted August 19, 2022

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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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KEY WORDS: steroidogenic acute regulatory protein, STARD1, Escherichia coli, cholesterol, biotransformation of steroids, fluorescence

DOI: 10.1134/S0006297922090127

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