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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]

Abstract: The emergence of bacterial resistance to common antibiotics poses a threat to human health and has rekindled an interest in antimicrobial peptides (AMPs). LHP7, a novel hybrid AMP containing 83 amino acid residues was designed on the basis of the LH28 and plectasin. LHP7 was expressed in Pichia pastoris, the total concentration of secreted protein reached 0.906g/L after 108h of methanol induction. Its antimicrobial activity was higher than that of the parent AMPs; the minimal inhibitory concentrations (MICs) of LHP7 against Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus suis were 0.091, 0.023 and 0.18μM, respectively. The antibacterial activity of LHP7 against clinical MRSA isolates (MICs=0.73–2.91μM) was enhanced over that of plectasin. The fractional inhibitory concentration (FIC) indicated a synergistic effect between LHP7 and ampicillin against MRSA (FIC=0.375), and combinations of LHP7 with gentamicin, rifampin or tetracycline provided evidence of additive effects (FIC=0.625–1.0). LHP7 exhibited a broad range of pH stability and thermostability, and a hemolytic activity of less than 5% below a concentration of 500μg/mL. It was resistant to pepsin and papain digestion, but sensitive to trypsin digestion. These results suggest that LHP7 might have potential as a broadly applied and clinically useful antimicrobial agent. [Copyright &y& Elsevier]

Plectasin, the first fungus-derived defensin from Pseudoplectania nigrella , is considered to be one of the most likely substitutes for antibiotics used to cure Staphyloccocus and Streptococcus infections. Recombinant expression of plectasin in Pichia pastoris X-33 was achieved. In this work, we aimed to develop a multi-copy gene cassette to enhance plectasin expression in P. pastoris . Expression vectors pPICZαA-Pn ( n = 1, 2, 4, or 8) harboring a one, two, four, or eight-copy gene cassette were constructed and confirmed by digestion with Bam HI and Bgl II. Vectors linearized by Bgl II were transformed into P. pastoris by electroporation. The gene copy number of the recombinants was quantified by real-time quantitative PCR. Yields of recombinant plectasin were 296, 409, 516, and 879 mg/L for the 1-, 2-, 4-, and 8-copy transformants, respectively, in a 5-L fermentor, demonstrating that an increase in copy number results in a proportional elevation in the expression level of recombinant plectasin. The purity of plectasin was 90.8%, and the yield was found to be 814 mg/L using ion-exchange chromatography. Multi-copy gene dosage enhances plectasin expression levels and highly reduces its production cost. Additionally, the above results could be helpful in improving the application and understanding of the P. pastoris expression system. [ABSTRACT FROM AUTHOR]

Highlights: [•] First report of cloning of a novel AP gene from Saccharomyces boulardii. [•] Heterologous expression of high-activity rAP in Pichia pastoris X-33. [•] rAP possess favorable enzymatic characters. [•] rAP possess the function of dephosphorylation of LPS. [•] rAP alleviate inflammation responses caused by LPS. [Copyright &y& Elsevier]