Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology

• Published in: Applied microbiology and biotechnology 2014-04, Vol.98 (8), p.3651-3658
• Main Authors: Luan, Chao, Zhang, Hai Wen, Song, De Guang, Xie, Yong Gang, Feng, Jie, Wang, Yi Zhen
• Format: Article
• Language: English
• Subjects: Acne; Animals; Antimicrobial agents; Antimicrobial peptides; Bacillus subtilis; Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Biomedical and Life Sciences; Biosynthesis; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Bungarus fasciatus; Cathelicidins - biosynthesis; Cathelicidins - genetics; Cation exchange; Chromatography; Chromatography, Affinity; Drug resistance; E coli; Endotoxins; Enzymes; Escherichia coli; Feed science; Gene Expression; Gram-positive bacteria; Hydrolysis; Laboratories; Life Sciences; Metabolic Engineering - methods; Microbial Genetics and Genomics; Microbiological research; Microbiology; Peptides; Physiological aspects; Properties; Proteins; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Studies; SUMO-1 Protein - biosynthesis; SUMO-1 Protein - genetics
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-013-5246-6

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.