title: Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli.
abstract: 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.
authors.affiliation: Department of Biology, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Katia
authors.initials: K
authors.lastname: Pane
authors.affiliation: Department of Biology, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Durante
authors.affiliation: Department of Biology, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Elio
authors.initials: E
authors.lastname: Pizzo
authors.affiliation: Department of Biology, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Varcamonti
authors.affiliation: Department of Biology, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Zanfardino
authors.affiliation: Department of Biology, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Sgambati
authors.affiliation: Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Seconda Università di Napoli, Caserta, Italy.
authors.forename: Antimo
authors.initials: A
authors.lastname: Di Maro
authors.affiliation: Department of Chemical Sciences, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Carpentieri
authors.affiliation: Department of Medicine and Surgery, Università degli Studi di Salerno, Baronissi, Italy.
authors.forename: Viviana
authors.initials: V
authors.lastname: Izzo
authors.affiliation: Department of Biology, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Di Donato
authors.affiliation: Department of Biology, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Cafaro
authors.affiliation: Department of Biology, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Notomista
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000378:metabolism*; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism
chemical_list: D023181:Antimicrobial Cationic Peptides; D002352:Carrier Proteins; D011994:Recombinant Proteins; D012260:Ribonucleases; C058703:ranpirnase