Recombinant expression of antimicrobial peptides using a novel self-cleaving aggregation tag in Escherichia coli.

Antimicrobial peptides (AMPs) are part of the innate immune system of complex multicellular organisms. Despite the fact that AMPs show great potential as a novel class of antibiotics, the lack of a cost-effective means for their mass production limits both basic research and clinical use. In this work, we describe a novel expression system for the production of antimicrobial peptides in Escherichia coli by combining ΔI-CM mini-intein with the self-assembling amphipathic peptide 18A to drive the formation of active aggregates. Two AMPs, human ß-defensin 2 and LL-37, were fused to the self-cleaving tag and expressed as active protein aggregates. The active aggregates were recovered by centrifugation and the intact antimicrobial peptides were released into solution by an intein-mediated cleavage reaction in cleaving buffer (phosphate-buffered saline supplemented with 40 mmol/L Bis-Tris, 2 mmol/L EDTA, pH 6.2). The peptides were further purified by cation-exchange chromatography. Peptides yields of 0.82 ± 0.24 and 0.59 ± 0.11 mg/L were achieved for human ß-defensin 2 and LL-37, respectively, with demonstrated antimicrobial activity. Using our expression system, intact antimicrobial peptides were recovered by simple centrifugation from active protein aggregates after the intein-mediated cleavage reaction. Thus, we provide an economical and efficient way to produce intact antimicrobial peptides in E. coli.

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

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.

High-yield soluble expression and simple purification of the antimicrobial peptide OG2 using the intein system in Escherichia coli.

OG2 is a modified antimicrobial peptide, that is, derived from the frog peptide Palustrin-OG1. It has high antimicrobial activity and low cytotoxicity, and it is therefore promising as a therapeutic agent. Both prokaryotic (Escherichia coli) and eukaryotic (Pichia pastoris) production host systems were used to produce OG2 in our previous study; however, it was difficult to achieve high expression yields and efficient purification. In this study, we achieved high-yield OG2 expression using the intein fusion system. The optimized OG2 gene was cloned into the pTWIN1 vector to generate pTWIN-OG2-intein2 (C-terminal fusion vector) and pTWIN-intein1-OG2 (N-terminal fusion vector). Nearly 70% of the expressed OG2-intein2 was soluble after the IPTG concentration and induction temperature were decreased, whereas only 42% of the expressed of intein1-OG2 was soluble. Up to 75 mg of OG2-intein2 was obtained from a 1l culture, and 85% of the protein was cleaved by 100 mM DTT. Intein1-OG2 was less amenable to cleavage due to the inhibition of cleavage by the N-terminal amino acid of OG2. The purified OG2 exhibited strong antimicrobial activity against E. coli K88. The intein system is the best currently available system for the cost-effective production of OG2.

Effects of thioredoxin: SUMO and intein on soluble fusion expression of an antimicrobial peptide OG2 in Escherichia coli.

OG2 is a modified antimicrobial peptide of Palustrin-OG1 (OG1), which is derived from Odorrana grahami frog. OG2 has shown much higher selective antimicrobial activity and lower hemolytic activity than OG1, indicating OG2 may be a promising antimicrobial agent. In this study, we investigated three fusion partners, including thioredoxin, Mxe GyrA intein, and small ubiquitin-like modifier (SUMO), each fused with OG2, and examined their effects on the expression level and solubility of OG2 in Escherichia coli. The codon-optimized OG2 gene was cloned into pET32a (+) and pTWIN1 for fusion with thioredoxin and Mxe GyrA intein, respectively. In addition, the SUMO-OG2 gene was amplified by splice overlap extension PCR method and was cloned into pET30a (+). All recombinant plasmids were then transformed into E. coli BL21(DE3)pLysS, and the expressed fusion proteins were verified. Upon isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction, OG2 fused with thioredoxin (Trx-OG2) showed the highest yield as a soluble fusion protein (50 mg/L), followed by Mxe GyrA intein (44 mg/L) and SUMO (11 mg/L). The thioredoxin-fused protein (Trx-OG2) was then purified by nickel-nitrilotriacetic acid chromatography and desalted by Sephadex G25. The OG2 released by both tobacco etch virus protease and enterokinase from Trx-OG2 showed strong antimicrobial activity against Staphylococcus aureus ATCC25923.