Preparation of Recombinant Formate Dehydrogenase from Thermotolerant Yeast Ogataea parapolymorpha and Crystallization of Its Apo- and Holo- Forms

Pometun, A.A.; Boyko, K.M.; Zubanova, S.A.; Nikolaeva, A.Y.; Atroshenko, D.L.; Savin, S.S.; Tishkov, V.I.

Информация о цитировании статьи получена из Web of Science, Scopus
Статья опубликована в журнале из перечня ВАК
Статья опубликована в журнале из списка Web of Science и/или Scopus
Дата последнего поиска статьи во внешних источниках: 17 июня 2021 г.

Авторы: Pometun A.A., Boyko K.M., Zubanova S.A., Nikolaeva A.Yu, Atroshenko D.L., Savin S.S., Tishkov V.I.
Журнал: Moscow University Chemistry Bulletin
Том: 76
Номер: 1
Год издания: 2021
Издательство: Allerton Press Inc.
Местоположение издательства: United States
Первая страница: 49
Последняя страница: 55
DOI: 10.3103/S0027131421010120
Аннотация: NAD+-dependent formate dehydrogenase from thermotolerant yeast Ogataea parapolymorpha DL-1 (OpaFDH,EC 1.2.1.2) with the additional N-terminal Gly residue and its double mutant OpaFDH_ADwere overexpressed in E. coli cells. The enzyme yield was, respectively, 6000 and 6200 units per liter of the cultivation medium. Purified enzymes were obtained as homogeneous preparations with a yield of 62%. The purification procedure included ultrasonic cell disruption, heat treatment of cell free extracts at 55°C for 15 min, and hydrophobic chromatography on the Phenyl Sepharose Fast Flow. Crystallization experiments with wild-type OpaFDH resulted in the preparation of crystals of the apo-form but not the holo-form. Crystals of the holo-form were prepared in the case of the OpaFDH_AD mutant in the presence of 7 mM NAD+ and 10 mM sodium azide. The size and quality of the crystals are sufficient to collect X-ray diffraction data and determine the enzyme three-dimensional structure.
Добавил в систему: Тишков Владимир Иванович

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Preparation of Recombinant Formate Dehydrogenase from Thermotolerant Yeast Ogataea parapolymorpha and Crystallization of Its Apo- and Holo- Formsстатья

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