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NZ17074, a member of the arenicin family from Arenicola marina , exhibited potent antimicrobial activity against bacteria and fungi, but had some toxicity to human erythrocytes and porcine intestinal epithelial cells. To resolve this problem, one novel peptide N2 was designed based on NZ17074 by replacing Gly in position 1, 12 with Ala. N2 exhibited a typical β-sheet conformation in aqueous and almost had no hemolysis. N2 was expressed in Pichia pastoris using the small ubiquitin-like modifier fusion technology. The yield of N2 was approximately 6.5 mg/l, and its molecular weight was 2665.0 Da. The minimum inhibitory concentrations of N2 against Gram-negative bacteria ranged from 0.125 to 4 μg/ml. The antibacterial activity of N2 against Escherichia coli CVCC 195 and Salmonella enteritidis CVCC 3377 was enhanced up to 1–3-fold over parent NZ17074. This result provides some information that assists in the rational design of toxic antimicrobial peptides. [ABSTRACT FROM AUTHOR]

As a promising antibiotic candidate, it is necessary to find a safe method to produce the porcine cathelicidin antimicrobial peptide PR39. In this study, the antimicrobial peptide PR39 was successfully expressed with the maltose-inducible vector pGJ148 in the Bacillus subtilis WB800N. After induction with 3% maltose concentration, the fusion protein was successfully secreted onto the culture medium directly. After purification a Ni–NTA resin column, the fusion protein with a concentration of 17 mg/l was obtained from fermentation culture. Then, the purified fusion protein was cleaved using enterokinase, and tag-free PR39 was released. Approximately 2 mg of the recombinant PR39 with a purity of approximately 92.0% was obtained from a 1-l culture medium after purification with cation exchange column. The evaluation of antimicrobial and haemolytic activity demonstrated that the recombinant PR39 exhibited a high antimicrobial activity against several Gram-negative strains and a low haemolytic activity (256 μM) against human red blood cells. The synthetic PR39 produced similar results. This work expanded and provided a safe protocol for the large-scale production of antimicrobial peptides in B. subtilis . [ABSTRACT FROM AUTHOR]

MP1106 is a novel variant of plectasin with potent activity against some Gram-positive pathogenic bacteria, especially for Streptococcus suis and Staphylococcus aureus . MP1106 was successfully expressed in Pichia pastoris X-33 using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The total protein concentration and antimicrobial activity were 998.3 mg/L and 51,200 AU/mL, respectively. After purification by cation exchange chromatography, recombinant MP1106 (rMP1106) with a concentration of 100.13 mg/L and a purity of 95.2% was obtained from fermentation culture. Its antimicrobial spectrum was similar to that of plectasin, and the antibacterial activity against S. suis CVCC3309 was enhanced up to 29-fold over that of plectasin. In addition, 97.9% of the S. suis CVCC606 bacteria were killed by 16× minimal inhibitory concentration (MIC) rMP1106 within 6 h. The percentage hemolysis by rMP1106 against mouse erythrocytes was 3.8% at a concentration of 512 μg/mL. Exposure of porcine intestinal epithelial cells to 16–128 μg/mL rMP1106 caused a 9.5% decrease in cell viability. The rMP1106 was stable over broad ranges of temperature, pH and NaCl concentration. In addition, rMP1106 was highly resistant to papain, proteinase K and snailase digestion but susceptible to trypsin and slightly susceptible to pepsin. These results suggested that rMP1106 has potential as a promising therapeutic drug against S. suis infections. [ABSTRACT FROM AUTHOR]