Hybrid proteins containing proteorhodopsin from Exiguobacterium sibiricum

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Abstract

The genes of hybrid proteins including Exiguobacterium sibiricum proteorhodopsin (ESR) and various N-terminal soluble domains have been constructed. Effective synthesis in Escherichia coli cells was observed only in the case of hybrids with chaperone Caf1M and maltose-binding protein MBP expressed as precursors with their own signal sequences. The study of the isolated MBP-ESR protein in micelles and proteoliposomes demonstrated formation and decay of the main photocycle intermediates at pH > 8. The photoelectric response of the hybrid proteins Caf-ESR and MBP-ESR is comparable in amplitude to the wild-type ESR response, indicating their homogeneous orientation in the membrane. The obtained constructions can be used to create bacterial expression systems for various retinal proteins, ensuring their uniform incorporation into proteoliposomes.

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L. E. Petrovskaya

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, RAS; Moscow Institute of Physics and Technology

Author for correspondence.
Email: lpetr65@yahoo.com
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; Institutskiy per. 9, Dolgoprudny, Moscow Region, 141701

E. A. Kryukova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, RAS; Emanuel Institute of Biochemical Physics

Email: lpetr65@yahoo.com
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; ul. Kosygina 4, Moscow, 119334

V. A. Bolshakov

Lomonosov Moscow State University

Email: lpetr65@yahoo.com

Faculty of Biology

Russian Federation, Leninskie gory 1/11, Moscow, 119234

E. P. Lukashev

Lomonosov Moscow State University

Email: lpetr65@yahoo.com

Faculty of Biology

Russian Federation, Leninskie gory 1/11, Moscow, 119234

S. A. Siletsky

Lomonosov Moscow State University

Email: lpetr65@yahoo.com

Belozersky Institute of Physico-Chemical Biology

Russian Federation, Leninskye gory 1/40, Moscow, 119992

M. D. Mamedov

Lomonosov Moscow State University

Email: lpetr65@yahoo.com

Belozersky Institute of Physico-Chemical Biology

Russian Federation, Leninskye gory 1/40, Moscow, 119992

R. V. Sudakov

Lomonosov Moscow State University

Email: lpetr65@yahoo.com

Belozersky Institute of Physico-Chemical Biology

Russian Federation, Leninskye gory 1/40, Moscow, 119992

D. A. Dolgikh

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, RAS; Emanuel Institute of Biochemical Physics; Lomonosov Moscow State University

Email: lpetr65@yahoo.com

Faculty of Biology, Lomonosov Moscow State University

Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; ul. Kosygina 4, Moscow, 119334; Leninskie gory 1/11, Moscow, 119234

M. P. Kirpichnikov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, RAS; Lomonosov Moscow State University

Email: lpetr65@yahoo.com

Faculty of Biology, Lomonosov Moscow State University

Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; Leninskie gory 1/11, Moscow, 119234

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Supplementary files

Supplementary Files
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2. Fig. 1. Schemes of the structure of hybrid proteins. SP is the signal sequence. The glycine-serine linker is shown in dark gray, and the hexahistidine sequence is shown in black.

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3. Fig. 2. Expression of ESR-containing fusion proteins in E. coli cells. Protein electrophoresis in 13% SDS-PAGE (a) and Western blot analysis (b) with anti-His-conjugate of membrane fraction samples from E. coli C43(DE3) cells expressing fusion proteins (lanes 1–7) and wild-type ESR (lane 8). Lanes 1–7 correspond to the numbering of the constructs in Fig. 1. M – protein molecular weight markers (kDa).

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4. Fig. 3. MBP-ESR photocycle in DDM micelles at pH 7 (a) and pH 9 (b).

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5. Fig. 4. Kinetics of transmembrane potential difference (ΔΨ) formation by proteoliposomes containing wild-type ESR and fusion proteins at pH 7.5. The arrow marks the moment of laser flash. Data for ESR, Caf-ESR, ESR-Cherry and ESR-Trx are taken from the article by Petrovskaya et al. [37].

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6. Fig. 5. Kinetics of light-induced changes in absorption at characteristic wavelengths by proteoliposomes containing MBP-ESR at pH 7.5 (a) and 8.5 (b).

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