title: A novel carrier molecule for high-level expression of peptide antibiotics in Escherichia coli.
abstract: Peptide antibiotics are often hard to express in engineered bacteria at high level. According to the properties of peptide antibiotics, a heterologous protein PaP3.30, encoded by ORF30 of Pseudomonas aeruginosa bacteriophage PaP3, was selected as a carrier molecule. The gene of the carrier molecule was constructed into the plasmid pQE-32 to give rise to the vector pQE-PaP30 for expression of peptide antibiotics in Escherichia coli. A his-tagged fusion protein was genetically constructed with a peptide antibiotic at its carboxy terminus. The novel carrier molecule was used for high-level expression of six peptide antibiotics with different sizes and isoelectric points in E. coli, which are hPAB-beta, MSI-78, Melletin, hBD-1, Cecropin A, and an ovine anion peptide. And further, one of six peptide antibiotics, hPAB-beta (an analog of a human peptide antibiotic), was taken as an example for studies of recovery of interesting products from the fusion partner, purification and antimicrobial activity evaluation. The results indicated that the expressed fusion protein existed as an inclusion body in the cytoplasm and the expression amounts of six peptide antibiotic fusions are all higher than 34% of the total cell protein. The expression products could be easily purified by Ni-NTA chromatography. Cyanogen bromide was used to cut at the methionine linker between the carrier and hPAB-beta peptide. hPAB-beta was recovered from the fusion partner and purified to homogeneity with High S cation-exchange and Bio-gel P6 gel chromatography. The bactericidal activities of the purified recombinant hPAB-beta against P. aeruginosa are 31-64 microg/ml, and against Staphylococcus aureus are > or = 128 microg/ml, being comparable to that of the chemical synthesis peptide. These results show that the carrier molecule can result in high-level expression of peptide antibiotics, and expression products can be easily recovered from their fusion partner and retain their bioactivity.
authors.affiliation: Department of Microbiology, College of Medicine, The Third Military Medical University/Key Lab of Microbial Engineering Under the Educational Committee in Chongqing, 400038, PR China.
authors.forename: Xian C
authors.initials: XC
authors.lastname: Rao
authors.forename: Shu
authors.initials: S
authors.lastname: Li
authors.forename: Jin C
authors.initials: JC
authors.lastname: Hu
authors.forename: Xiao L
authors.initials: XL
authors.lastname: Jin
authors.forename: Xiao M
authors.initials: XM
authors.lastname: Hu
authors.forename: Jian J
authors.initials: JJ
authors.lastname: Huang
authors.forename: Zi J
authors.initials: ZJ
authors.lastname: Chen
authors.forename: Jun M
authors.initials: JM
authors.lastname: Zhu
authors.forename: Fu Q
authors.initials: FQ
authors.lastname: Hu
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides; D015202:Protein Engineering; D017105:Pseudomonas Phages / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000821:virology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013211:Staphylococcus aureus / Q000187:drug effects; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins