title: Application of immobilized thrombin for production of S-thanatin expressed in Escherichia coli.
abstract: S-thanatin, a small antimicrobial peptide with 21 amino acid residues, was expressed as a fusion protein containing thrombin cleavage site in Escherichia coli BL21 (DE3). To reduce the production cost, immobilization of thrombin in polyacrylamide gel for cleavage was studied in this work. The immobilized thrombin exhibited excellent activity within wider ranges of pH value and temperature for reaction than free enzyme, and the residual activity could remain above 75% after ten times of usage. Tricine-SDS-PAGE result showed that the immobilized thrombin could cleave the S-thanatin fusion protein effectively. After cleavage, recombinant S-thanatin was purified by preparative reversed-phase high-performance liquid chromatography and mass spectrum showed that the molecular weight (2,448.86) was close to the theoretical value (2,448.98). After purification, about 7 mg of S-thanatin was obtained from 1 l of culture and the recombinant exhibited excellent bioactivity to E. coli ATCC 25922, with the minimum inhibitory concentration of 12 μg/ml. The purification method could be applied to prepare other peptides with similar properties at low cost.
authors.affiliation: Center of Clinical Laboratory Medicine of Zhongda Hospital, Southeast University, Nanjing, China. nationball@163.com
authors.forename: Guoqiu
authors.initials: G
authors.lastname: Wu
authors.forename: Xuepeng
authors.initials: X
authors.lastname: Deng
authors.forename: Xiaofang
authors.initials: X
authors.lastname: Li
authors.forename: Xiyong
authors.initials: X
authors.lastname: Wang
authors.forename: Shenglan
authors.initials: S
authors.lastname: Wang
authors.forename: Hanmei
authors.initials: H
authors.lastname: Xu
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008826:Microbial Sensitivity Tests; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D023181:Antimicrobial Cationic Peptides; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; C098076:thanatin; D013917:Thrombin