title: Recombinant expression, purification and antimicrobial activity of a novel antimicrobial peptide PaDef in Pichia pastoris.
abstract: The antimicrobial peptide PaDef was isolated from Mexican avocado fruit and was reported to inhibit the growth of Escherichia coli and Staphylococcus aureus in 2013. In this study, an N-terminal 6 × His tagged recombinant PaDef (rPaDef) with a molecular weight of 7.5 KDa, for the first time, was expressed as a secreted peptide in Pichia pastoris. The optimal culture condition for rPaDef expression was determined to be incubation with 1.5% methanol for 72 h at 28 °C under pH 6.0. Under this condition, the amount of the rPaDef accumulation reached as high as 79.6 μg per 1 ml of culture medium. Once the rPaDef peptide was purified to reach a 95.7% purity using one-step nickel affinity chromatography, its strong and concentration-dependent antimicrobial activity was detected to be against a broad-spectrum of bacteria of both Gram-negative and Gram-positive. The growth of these bacterial pathogens was almost completely inhibited when the rPaDef peptide was at a concentration of as low as 90 μg/ml. In summary, our data showed that rPaDef derived from Mexican avocado fruit can be expressed and secreted efficiently when P. pastoris was used as a cell factory. This is the first report on heterologous expression of PaDef in P. pastoris and the approach described holds great promise for antibacterial drug development.
authors.affiliation: China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
authors.forename: De-Mei
authors.initials: DM
authors.lastname: Meng
authors.affiliation: China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Jing-Fang
authors.initials: JF
authors.lastname: Zhao
authors.affiliation: China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Ling
authors.affiliation: China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Hong-Xia
authors.initials: HX
authors.lastname: Dai
authors.affiliation: China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Ya-Jun
authors.initials: YJ
authors.lastname: Guo
authors.affiliation: China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Xiao-Fang
authors.initials: XF
authors.lastname: Gao
authors.affiliation: China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Bin
authors.initials: B
authors.lastname: Dong
authors.affiliation: China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Zi-Qi
authors.initials: ZQ
authors.lastname: Zhang
authors.affiliation: College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Xin
authors.initials: X
authors.lastname: Meng
authors.affiliation: China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China; Tianjin Food Safety & Low Carbon Manufacturing Collaborative Innovation Center, Tianjin, 300457, People's Republic of China; Obesita & Algaegen LLC, College Station, TX, 77845, USA. Electronic address: fanzhen@tust.edu.cn.
authors.forename: Zhen-Chuan
authors.initials: ZC
authors.lastname: Fan
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D004926:Escherichia coli / Q000254:growth & development*; D027421:Persea / Q000737:chemistry / Q000235:genetics*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000254:growth & development*
chemical_list: D023181:Antimicrobial Cationic Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins
keywords: Antibacterial activity; Antimicrobial peptide; PaDef; Pichia pastoris; Secreted expression