title: 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.
abstract: 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 1L of culture (20 g wet cells). BSAP shows 515 U/mg activity at optimum conditions (pH 11 and 0.8-2M 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.
authors.affiliation: Group of Reagents Technologies, Research Center of Biomedicine and Health, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China. mkoksharov@gmail.com
authors.forename: Mikhail
authors.initials: M
authors.lastname: Koksharov
authors.forename: Changqing
authors.initials: C
authors.lastname: Lv
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Zhai
authors.forename: Natalia
authors.initials: N
authors.lastname: Ugarova
authors.forename: Eric
authors.initials: E
authors.lastname: Huang
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D019897:Periplasm / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase
keywords: Alkaline phosphatase; Bacillus subtilis; Expression in Escherichia coli; Protein folding; Solubility; Stationary phase