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Expression of Xanthorhodopsin in Escherichia coli

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Abstract

Xanthorhodopsin (XR) from Salinibacter ruber is a light-driven proton pump containing retinal and a light-harvesting carotenoid antenna salinixanthin. Previous structure-functional studies of XR were conducted using a protein isolated from the native host only due to the absence of heterologous expression in Escherichia coli. In this paper, we describe cell-free synthesis and incorporation in lipid–protein nanodiscs of the recombinant XR that demonstrated its principal compatibility with E. coli biosynthetic machinery. To produce XR in E. coli, three C-terminal deletion variants of this protein were constructed. In contrast to the full-length XR, their expression resulted in efficient synthesis in E. coli cells. However, cells producing recombinant XR variants bound retinal only upon growth in minimal medium, not in the rich one. The XR3 variant with deletion of ten C-terminal amino acid residues was obtained and characterized. Its absorption spectrum and photocycle kinetics were close to those reported for XR isolated from S. ruber membranes and bleached from salinixanthin. We have also constructed the first mutants of XR, H62M and D96N, and examined their properties.

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Data Availability

The data are available from the corresponding authors on reasonable request.

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Acknowledgements

We thank Mrs. Jennifer M. Wang for her excellent technical assistance.

Funding

The study was financially supported by the Ministry of Science and Higher Education of the Russian Federation (project no. 075-15-2021-1354 from 07.10.2021) (protein production and spectroscopic studies), Russian Scientific Foundation Grant No. 22-14-00104 (mutant genes construction), and, at early stages, by grants from NIH (GM29498), DOE (DEFG03-86ER13525) to J.K.L., and ARO (W911NF-06-01-0020 and W911NF-09-1-0243) to S.P.B. and J.K.L. (gene cloning and experiments with SX).

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Authors and Affiliations

  1. Laboratory of Protein Engineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, ul. Miklukho-Maklaya, 16/10, Moscow, 117997, Russia

    Lada E. Petrovskaya & Dmitriy A. Dolgikh

  2. Department of Biology, Lomonosov Moscow State University, Moscow, 119991, Russia

    Evgeniy P. Lukashev, Dmitriy A. Dolgikh, Evgeniy G. Maksimov, Andrei B. Rubin & Mikhail P. Kirpichnikov

  3. Faculty of Biology, Shenzhen MSU-BIT University, Shenzhen, 518172, China

    Ekaterina N. Lyukmanova & Mikhail A. Shulepko

  4. Laboratory of Bioengineering of Neuroreceptors and Neuromodulators, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, 117997, Russia

    Ekaterina N. Lyukmanova & Mikhail A. Shulepko

  5. Group of Nanobioengineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, 117997, Russia

    Elena A. Kryukova

  6. Group of Mass Spectrometry, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia

    Rustam H. Ziganshin

  7. Department of Bioengineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia

    Mikhail P. Kirpichnikov

  8. Department of Physiology and Biophysics, D-320, Medical Sciences I, University of California, Irvine, CA, 92697-4560, USA

    Janos K. Lanyi & Sergei P. Balashov

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  1. Lada E. Petrovskaya
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  2. Evgeniy P. Lukashev
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  3. Ekaterina N. Lyukmanova
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  7. Dmitriy A. Dolgikh
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  8. Evgeniy G. Maksimov
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  11. Janos K. Lanyi
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  12. Sergei P. Balashov
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Contributions

LEP, EPL and SPB designed the research and analyzed data. LEP, EPL, RHZ, EAK, ENL, and MAS carried out the experiments. EGM and SPB advised and supervised measurements. Project administration, EGM, MPK, ABR, JKL and SPB. LEP, EPL, ENL, DAD and SPB wrote the text. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Lada E. Petrovskaya or Sergei P. Balashov.

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The authors declare that they have no conflict of interest.

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Petrovskaya, L.E., Lukashev, E.P., Lyukmanova, E.N. et al. Expression of Xanthorhodopsin in Escherichia coli. Protein J 42, 408–420 (2023). https://doi.org/10.1007/s10930-023-10109-5

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