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.

Expression of plectasin in Bacillus subtilis using SUMO technology by a maltose-inducible vector

“…Usually, an N-terminal 6 × His-tag is fused to SUMO for Ni-NTA affinity purification to obtain suitable purity of the target fusion protein. Now, the SUMO fusion technology has been proved to be an effective expression tool of AMPs [3,11,13]. In our work, a 6 × His-tag and yeast SUMO (Saccharomyces cerevisiae Smt3) were fused to the N-terminal of plectasin.…”

“…Nowadays, isopropyl-β-D-thiogalactopyranoside (IPTG) induction promoter Pspac has been widely used in the expression of AMPs in B. subtilis [8,13]. However, IPTG is expensive and toxicity, which determines that Pspac is not an ideal choice for an expression system.…”

Considerations for the process development of insect‐derived antimicrobial peptide production

“…For example the expression of A3APO‐ and Alyteserin, both toxic for E. coli and Salmonella strains, was produced in Lactococcus lactis . Cathelicidin‐BF showing toxicity against gram‐negative and gram‐positive bacteria was produced in Bacillus subtilis …”

“…31 Cathelicidin-BF showing toxicity against gram-negative and gram-positive bacteria was produced in Bacillus subtilis. 32 An alternative yeast expression organism for the production of AMPs is Kluyveromyces lactis (K. lactis) which has been already used for the recombinant production of AMPs like the Saposin-Like Domain of the Aspartic Protease Cirsin. 33 The advantages of K. lactis such as simple genetic manipulation, the ability to use both integrative and episomal expression vectors, and the availability of a fully sequenced genome made it an attractive alternative to P. pastoris.…”

Hitchhiking with Nature: Snake Venom Peptides to Fight Cancer and Superbugs

“…Both systems retained the antibacterial activity of the free antimicrobial peptide and released Bf-CATH over >15 days [128,129]. Different recombinant DNA strategies were also reported to effectively produce Bf-CATH, such as small ubiquitin-related modifier (SUMO) technology or intein-based technology, both expressed in Bacillus subtilis (achieved peptide yields of~3 mg/L and 0.5 mg/L, respectively) [130,131].…”