title: Expression, purification and initial characterization of a novel recombinant antimicrobial peptide Mytichitin-A in Pichia pastoris.
abstract: Mytichitin-A is an antimicrobial peptide isolated from the serum of Mytilus coruscus and is reported to inhibit bacterial growth as tested on several Gram-positive bacteria. To produce large quantity of Mytichitin-A to further investigate its biological activity, nucleotide sequence encoding a recombinant 6 × His-Mytichitin-A (rMytichitin-A) peptide was synthesized and inserted into the inducible yeast expression vector pPICZαA. With the availability of such an expression vector called pPICZαA-Mytichitin-A, we transformed Pichia pastoris GS115 cells with a SacI-linearized pPICZαA-Mytichitin-A by electroporation. Transgenic strains secreting rMytichitin-A with a molecular weight of approximate 10 KDa as expected were obtained. The optimal culture condition for rMytichitin-A expression was determined to be 1.0% methanol induction, 96 h incubation at 28 °C and the amount of rMytichitin-A reached 45.5 μg/ml. The percentage of rMytichitin-A was estimated to be 73.6% of the total protein. After rMytichitin-A was purified using nickel ions affinity chromatography, approximate 9.1 mg pure rMytichitin-A was obtained from 500 ml of cell culture medium with 97.8% purity. More importantly, both the culture supernatant and purified rMytichitin-A inhibited the growth of Gram-positive bacteria, especially Staphylococcus aureus and Bacillus subtilis with a minimum inhibition concentration of as low as 31 and 48 μg/ml, respectively. Differently from the native protein, however, the rMytichitin-A is not active against Gram-negative bacteria. Taken together, this is the first report on the heterologous expression of Mytichitin-A in P. pastoris. Our study showed that P. pastoris is an effective expression system for producing large quantities of biologically active Mytichitin-A for both research and application purposes.
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: 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: 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: 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: 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
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: 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: 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, United States. Electronic address: fanzhen@tust.edu.cn.
authors.forename: Zhen-Chuan
authors.initials: ZC
authors.lastname: Fan
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D001412:Bacillus subtilis / Q000254:growth & development*; D006090:Gram-Negative Bacteria / Q000254:growth & development*; D049878:Mytilus / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000493:pharmacokinetics; D013211:Staphylococcus aureus / Q000254:growth & development*
chemical_list: D023181:Antimicrobial Cationic Peptides; D011994:Recombinant Proteins
keywords: Antimicrobial activity; Antimicrobial peptide; Expression; Pichia pastoris; Recombinant Mytichitin-A