Expression, purification and antimicrobial activity analysis of recombination peptide subtilosin A in Escherichia coli using SUMO fusion technology

Abstract

Subtilosin A is an antimicrobial peptide isolated from Bacillus subtilis, and it possess broad-spectrum antibacterial activity. As subtilosin A prevents biofilm formation by inhibiting bacterial quorum sensing processes against Gram-positive, Gram-negative, and Gram-variable bacteria, it is urgent to obtain high-quality subtilosin A production in an economical and effective way. Escherichia coli is undoubtedly a most preferred host system for production of heterologous recombinant proteins. However, subtilosin A has the inhibitory effects against E. coli and it is easily to be degraded. To produce subtilosin A in E. coli, the application of small ubiquitin-related modifier (SUMO) fusion system with the purpose of stability and prevention of antimicrobial activity is the best practices in this regard. In this study, subtilosin A gene with codon optimization was cloned into the Stu I/Hind III sites of pSUMO vector after SUMO tag and transformed to E. coli BL21 (DE3). The SUMO-subtilosin A fusion protein was induced at 15ºC for 16 hours with 0.5 mM isopropyl thio-β-D- galactoside (IPTG) induction. The fusion protein with a molecular weight of approximately 20 kDa was confirmed by SDS-PAGE. The subtilosin A was then released by SUMO protease cleavage at the junction site and isolated and purified by affinity and cation exchange chromatography. The recombinant subtilosin A demonstrated a good antibacterial activity against Pseudomonas aeruginosa (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria), with minimum inhibitory concentrations (MICs) of 100 mg/L and 100 mg/L, respectively. These results showed an efficient method for synthesis functional subtilosin A.