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.
Supplemental Figure S1. Toxicity of diquat in mice. Thirty mice (five-week-old) were chosen to establish the mouse model of intestinal oxidative stress. After one week of adaption, these mice were randomly allocated into six groups (n=5) and treated as follows: (a) PBS (control group), (b) 12 mg/kg diquat group, (c) 25 mg/kg diquat group, (d) 50 mg/kg diquat group, (e) 75 mg/kg diquat group, (f) 100 mg/kg diquat group. Equal volumes of diquat or PBS was injected intraperitoneally. Twelve hours after treatment, the mice that survived were counted and samples of their blood, liver, duodenum, jejunum, ileum and colon were collected.
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