title: Expressing antimicrobial peptide cathelicidin-BF in Bacillus  subtilis using SUMO technology.
abstract: Small ubiquitin-related modifier (SUMO) technology has been widely used in Escherichia coli expression systems to produce antimicrobial peptides. However, E. coli is a pathogenic bacterium that produces endotoxins and can secrete proteins into the periplasm, forming inclusion bodies. In our work, cathelicidin-BF (CBF), an antimicrobial peptide purified from Bungarus fasciatus venom, was produced in a Bacillus subtilis expression system using SUMO technology. The chimeric genes his-SUMO-CBF and his-SUMO protease 1 were ligated into vector pHT43 and expressed in B. subtilis WB800N. Approximately 22 mg of recombinant fusion protein SUMO-CBF and 1 mg of SUMO protease 1 were purified per liter of culture supernatant. Purified SUMO protease 1 was highly active and cleaved his-SUMO-CBF with an enzyme-to-substrate ratio of 1:40. Following cleavage, recombinant CBF was further purified by affinity and cation exchange chromatography. Peptide yields of ~3 mg/l endotoxin-free CBF were achieved, and the peptide demonstrated antimicrobial activity. This is the first report of the production of an endotoxin-free antimicrobial peptide, CBF, by recombinant DNA technology, as well as the first time purified SUMO protease 1 with high activity has been produced from B. subtilis. This work has expanded the application of SUMO fusion technology and may represent a safe and efficient way to generate peptides and proteins in B. subtilis.
authors.forename: Chao
authors.initials: C
authors.lastname: Luan
authors.forename: Hai Wen
authors.initials: HW
authors.lastname: Zhang
authors.forename: De Guang
authors.initials: DG
authors.lastname: Song
authors.forename: Yong Gang
authors.initials: YG
authors.lastname: Xie
authors.forename: Jie
authors.initials: J
authors.lastname: Feng
authors.forename: Yi Zhen
authors.initials: YZ
authors.lastname: Wang
mesh_terms: D000818:Animals; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D054804:Cathelicidins / Q000096:biosynthesis* / Q000235:genetics*; D002846:Chromatography, Affinity; D015870:Gene Expression; D006868:Hydrolysis; D060847:Metabolic Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D025842:SUMO-1 Protein / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D054804:Cathelicidins; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; C533044:cathelicidin BF, Bungarus fasciatus