Journal
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
Volume 67, Issue 35, Pages 9749-9756Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.9b02350
Keywords
bovine lactoferrin N-lobe; Bacillus subtilis; expression; codon bias; promoter optimization
Funding
- National Key research and Development Program of China [SQ2018YFA090039-02]
- Program for Advanced Talents within Six Industries of Jiangsu Province [2015-SWYY-010]
- National First-class Discipline Program of Light Industry Technology and Engineering [LITE2018-12]
- Program of Introducing Talents of Discipline to Universities [111-2-06]
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Bovine lactoferrin N-lobe plays an important key in the nonimmunological defense system. In this work, the most suitable promoter P-veg was selected and the fragment coding bovine lactoferrin N-lobe was optimized according to codon bias of Bacillus. The recombinant plasmid pMA0911-P-veg-mBLF-N was introduced into Baicillus subtilis 168 to create B. subtilis/pMA0911-P-veg-mBLF-N. The bovine lactoferrin N-lobe was highly expressed at 28 degrees C for 15 h. Its purified protein was obtained with 16.5 mg/L and a purity of 93.6% using ammonium sulfate precipitation, Ni-NTA, and molecular exclusion. About 200 ng/mL purified bovine lactoferrin N-lobe completely inhibited cell-growth of Escherichia coli JM109 (DE3), 70.3% of Pseudomonas aeruginosa CGMCC 1.6740, and 41.5% of Staphylococcus aureus CGMCC 1.282. To our knowledge, this is the first report about active expression, purification, and characterization of bovine lactoferrin N-lobe in safe bacterium B. subtilis, which opens an available application way in the biomedical and food industries.
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