Development of a Bacterial Expression System for Producing 15N/13C-Labeled Neuroglobin and Cytochrome C

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Abstract

An effective system for producing isotopically labeled heme-containing proteins of human neuroglobin and cytochrome c has been developed. The corresponding producer strains have been obtained, and optimal cultivation conditions have been selected: temperature and duration of incubation, composition of media, and concentration of expression inducer. Using far- and near-UV-vis CD spectroscopy, it was shown that the secondary structure composition of 15N-neuroglobin is close to the calculated one, and the orientation of the heme in the molecules is predominantly canonical. According to two-dimensional 1H/15N-HSQC NMR spectra of human neuroglobin and cytochrome c, the proteins are folded into a native conformation with a predominance of the α-helical structure. The resulting production system can be used to produce highly purified preparations of totally 15N/13C-labeled proteins for structural-dynamic studies using modern high-resolution NMR spectroscopy methods.

About the authors

M. A. Semenova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS

Moscow, Russia

O. M. Smirnova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS

Moscow, Russia

V. V. Britikov

Institute of Bioorganic Chemistry, NAS of Belarus

Minsk, Belarus

E. V. Britikova

Institute of Bioorganic Chemistry, NAS of Belarus

Minsk, Belarus

A. P. Khodnenko

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS

Moscow, Russia

Y. V. Bershatsky

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS

Moscow, Russia

A. A. Ignatova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS

Moscow, Russia

E. V. Bocharov

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

Moscow, Russia; Dolgoprudny, Russia

M. P. Kirpichnikov

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS; Biological faculty, Lomonosov Moscow State University

Moscow, Russia; Moscow, Russia

D. A. Dolgikh

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS; Biological faculty, Lomonosov Moscow State University

Moscow, Russia; Moscow, Russia

R. V. Chertkova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS

Email: cherita@inbox.ru
Moscow, Russia

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