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

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Expression

Background:

• Small ubiquitin-related modifier (SUMO) technology is commonly used in Escherichia coli for antimicrobial peptide production.
• Escherichia coli poses risks due to endotoxins and inclusion body formation.
• Bacillus subtilis offers a safer alternative expression system.

Purpose of the Study:

• To produce endotoxin-free cathelicidin-BF (CBF) using SUMO technology in Bacillus subtilis.
• To develop a safe and efficient recombinant protein production system.
• To report the production of active SUMO protease 1 in B. subtilis.

Main Methods:

• Constructed chimeric genes (his-SUMO-CBF and his-SUMO protease 1) and ligated them into vector pHT43.
• Expressed genes in Bacillus subtilis WB800N.
• Purified SUMO-CBF fusion protein and SUMO protease 1 from culture supernatant.
• Cleaved SUMO-CBF with SUMO protease 1 and purified recombinant CBF using chromatography.

Main Results:

• Achieved yields of approximately 22 mg/L SUMO-CBF and 1 mg/L SUMO protease 1.
• Demonstrated high activity of purified SUMO protease 1, cleaving SUMO-CBF at a 1:40 ratio.
• Obtained ~3 mg/L of purified, endotoxin-free CBF with demonstrated antimicrobial activity.

Conclusions:

• Successfully produced endotoxin-free antimicrobial peptide CBF in Bacillus subtilis using SUMO technology.
• Reported the first production of highly active SUMO protease 1 from B. subtilis.
• Established a safe and efficient method for recombinant peptide and protein generation in B. subtilis, expanding SUMO fusion technology applications.