Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200–250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15–18 mg of recombinant peptide per liter of culture with 96–98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods.
Novel approaches are needed to combat antibiotic resistance. Here, we describe a computational-experimental framework for the discovery of novel cryptic antimicrobial peptides (AMPs). The computational platform, based on previously validated antimicrobial scoring functions, indicated the activation peptide of pepsin A, the main human stomach protease, and its N- and C-terminal halves as antimicrobial peptides. The three peptides from pepsinogen A3 isoform were prepared in a recombinant form using a fusion carrier specifically developed to express toxic peptides in Escherichia coli. Recombinant pepsinogen A3-derived peptides proved to be wide-spectrum antimicrobial agents with MIC values in the range 1.56-50 μM (1.56-12.5 μM for the whole activation peptide). Moreover, the activation peptide was bactericidal at pH 3.5 for relevant foodborne pathogens, suggesting that this new class of previously unexplored AMPs may contribute to microbial surveillance within the human stomach. The peptides showed no toxicity toward human cells and exhibited anti-infective activity in vivo, reducing by up to 4 orders of magnitude the bacterial load in a mouse skin infection model. These peptides thus represent a promising new class of antibiotics. We envision that computationally guided data mining approaches such as the one described here will lead to the discovery of antibiotics from previously unexplored sources.
Ribosome-inactivating proteins (RIPs) are enzymes (3.2.2.22) that possess N-glycosilase activity that irreversibly inhibits protein synthesis. RIPs have been found in plants, fungi, algae, and bacteria; their biological role is still under investigation, even if it has been recognized their role in plant defence against predators and viruses. Nevertheless, several studies on these toxins have been performed to evaluate their applicability in the biomedical field making RIPs selectively toxic towards target cells. Indeed, these molecules are extensively used to produce chimeric biomolecules, such as immunotoxins or protein/peptides conjugates. However, to date, clinical use of most of these bioconiujates has been limited by toxicity and immunogenicity. More recently, material sciences have provided a wide range of nanomaterials to be used as excellent vehicles for toxin-delivery, since they are characterized by improved stability, solubility, and in vivo pharmacokinetics. This review discusses progresses in the development of RIPs bioconjugates, with particular attention to the recent use of nanomaterials, whose appropriate design opens up a broad range of different possibilities to the use of RIPs in novel therapeutic approaches in human diseases.
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