High Level Expression and Purification of the Clinically Active Antimicrobial Peptide P-113 in Escherichia coli

P-113, which was originally derived from the human saliva protein histatin 5, is a histidine-rich antimicrobial peptide with the sequence AKRHHGYKRKFH. P-113 is currently undergoing phase II clinical trial as a pharmaceutical agent to fight against fungal infections in HIV patients with oral candidiasis. Previously, we developed a new procedure for the high-yield expression and purification of hG31P, an analogue and antagonist of human CXCL8. Moreover, we have successfully removed lipopolysaccharide (LPS, endotoxin) associated with hG31P in the expression with Escherichia coli. In this paper, we have used hG31P as a novel fusion protein for the expression and purification of P-113. The purity of the expressed P-113 is more than 95% and the yield is 4 mg P-113 per liter of E. coli cell culture in Luria-Bertani (LB) medium. The antimicrobial activity of the purified P-113 was tested. Furthermore, we used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to study the structural properties of P-113. Our results indicate that using hG31P as a fusion protein to obtain large quantities of P-113 is feasible and is easy to scale up for commercial production. An effective way of producing enough P-113 for future clinical studies is evident in this study

High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in <i>Escherichia coli</i>

Cecropins are a family of antimicrobial peptides (AMPs) that are widely found in the innate immune system of Cecropia moths. Cecropins exhibit a broad spectrum of antimicrobial and anticancer activities. The structures of Cecropins are composed of 34–39 amino acids with an N-terminal amphipathic α-helix, an AGP hinge and a hydrophobic C-terminal α-helix. KR12AGPWR6 was designed based on the Cecropin-like structural feature. In addition to its antimicrobial activities, KR12AGPWR6 also possesses enhanced salt resistance, antiendotoxin and anticancer properties. Herein, we have developed a strategy to produce recombinant KR12AGPWR6 through a salt-sensitive, pH and temperature dependent intein self-cleavage system. The His6-Intein-KR12AGPWR6 was expressed by E. coli and KR12AGPWR6 was released by the self-cleavage of intein under optimized ionic strength, pH and temperature conditions. The molecular weight and structural feature of the recombinant KR12AGPWR6 was determined by MALDI-TOF mass, CD, and NMR spectroscopy. The recombinant KR12AGPWR6 exhibited similar antimicrobial activities compared to the chemically synthesized KR12AGPWR6. Our results provide a potential strategy to obtain large quantities of AMPs and this method is feasible and easy to scale up for commercial production

Novel Antimicrobial Peptides with High Anticancer Activity and Selectivity

<div><p>We describe a strategy to boost anticancer activity and reduce normal cell toxicity of short antimicrobial peptides by adding positive charge amino acids and non-nature bulky amino acid β-naphthylalanine residues to their termini. Among the designed peptides, K4R2-Nal2-S1 displayed better salt resistance and less toxicity to hRBCs and human fibroblast than Nal2-S1 and K6-Nal2-S1. Fluorescence microscopic studies indicated that the FITC-labeled K4R2-Nal2-S1 preferentially binds cancer cells and causes apoptotic cell death. Moreover, a significant inhibition in human lung tumor growth was observed in the xenograft mice treated with K4R2-Nal2-S1. Our strategy provides new opportunities in the development of highly effective and selective antimicrobial and anticancer peptide-based therapeutics.</p></div

Hemolysis and cytotoxicity to human red blood cells (hRBCs) and human fibroblast (HFW) cells of S1, Nal2-S1, K4R2-Nal2-S1, and K6-Nal2-S1.

<p>Hemolysis and cytotoxicity to human red blood cells (hRBCs) and human fibroblast (HFW) cells of S1, Nal2-S1, K4R2-Nal2-S1, and K6-Nal2-S1.</p

K4R2-Nal2-S1 treatment attenuates xenograft tumor growth.

<p>(a) Dorsal sides of male nude mice s.c. injected with PC9 human lung cancer cells and i.v. treated with K4R2Nal2-S1 (right) or PBS control (left) at the 46^th day after cancer cell implantation (5 days for tumor growth, 40 days for treatment, photographed on the 46^th day). (b) Mice body weight (left) and tumor volume (right) from (a) were monitored over the time period as indicated. (c) Exposed tumors (mice were sacrificed at the 46^th day after cancer cell implantation) of mice treated with K4R2Nal2-S1 (upper) or PBS (lower). 12 exposed tumors were found in the PBS group (6 mice x 2 side implantation), but only 7 exposed tumors were found in the K4R2Nal2-S1 treatment group. Total tumor weight for both groups were measured and shown in right.</p

Excised xenografted tumors from the mice in Fig 3 were subjected to H&E staining and immunohistochemical assay for cleaved PARP expression to monitor cellular apopotosis.

<p>Scale bars = 500 μm, 100 μm, and 50 μm (40X, 200X, and 400X), respectively.</p

Plots of the activities of S1, Nal2-S1, K4R2-Nal2-S1, and K6-Nal2-S1 against OECM-1, C9, SAS, A549, PC9, PC9-G cancer cell lines.

<p>Plots of the activities of S1, Nal2-S1, K4R2-Nal2-S1, and K6-Nal2-S1 against OECM-1, C9, SAS, A549, PC9, PC9-G cancer cell lines.</p