Bacillus subtilis alkaline phosphatase IV acquires activity only late at the stationary phase when produced in Escherichia coli. Overexpression and characterization of the recombinant enzyme

Koksharov, M.; Changqing, L.; Zhai, X.; Ugarova, N.; Huang, E.

Авторы: Koksharov Mikhail, Changqing Lv, Zhai Xiaohong, Ugarova Natalia, Huang Eric
Журнал: Protein Expression and Purification
Том: 90
Номер: 2
Год издания: 2013
Издательство: Academic Press
Местоположение издательства: United States
Первая страница: 186
Последняя страница: 194
DOI: 10.1016/j.pep.2013.06.008
Аннотация: The availability of recombinant monomeric alkaline phosphatase (AP) is highly desirable in analytical applications involving AP fusion proteins. The cobalt-dependant alkaline phosphatase IV from Bacillus subtilis (BSAP), which was reported to be strongly monomeric, was overexpressed in Escherichia coli using pET autoinduction system as a cytoplasmic protein without export signal sequence. After 1 day of growth, when the E. coli culture was near the stationary phase (standard time to harvest protein in this expression method), high amounts of BSAP were produced but the soluble fraction of BSAP was nearly inactive: the AP activity in the cell-free extract was near the background level. However, further incubation of bacterial culture lead to a tremendous increase in AP activity which was maximal at the 3rd day of incubation and was 48-100 times higher than at the 1st day of growth. The recombinant BSAP was purified by metal-chelate chromatography and characterized. Typically, 90-140 mg of active protein was produced in 1 L of culture (20 g wet cells). BSAP shows 515 U/mg activity at optimum conditions (pH 11 and 0.8–2 M NaCl). Contrary to the previous report on the native enzyme, BSAP was found to be dimeric and showed only negligible diesterase activity. The observed unusual late activation of BSAP indicates that prolonged incubation at the stationary phase may be useful for functional expression of some problematic proteins in E. coli.
Добавил в систему: Кокшаров Михаил Иванович

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Bacillus subtilis alkaline phosphatase IV acquires activity only late at the stationary phase when produced in Escherichia coli. Overexpression and characterization of the recombinant enzymeстатья

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