title: Production of antifungal recombinant peptides in Escherichia coli.
abstract: Antifungal peptides derived from the human bactericidal/permeability-increasing protein (BPI) were produced in Escherichia coli as fusion proteins with human BoneD. Bacterial cultures transformed with the gene encoding the fusion protein were grown to a high cell density (OD(600)>100), and induced with L-arabinose to initiate product expression. Fusion protein accumulated into cytoplasmic inclusion bodies and recombinant peptide was released from BoneD by acid hydrolysis at an engineered aspartyl-prolyl dipeptide linker. Acid hydrolysis of purified inclusion bodies at pH <2.6 followed Arrhenius kinetics and did not require prior inclusion body solubilization in detergents or denaturants. Surprisingly, at pH <2.6 and 85 degrees C, cell lysis and aspartyl-prolyl hydrolysis with concomitant peptide release occurred simultaneously. Bacterial cultures were, therefore, adjusted to approximately pH 2.6 with HCl directly in the bioreactor and incubated at elevated temperature. Peptide, which is soluble in the aqueous acidic environment, was separated from the insoluble material and purified using column separation techniques. Recombinant peptide was separated from the hydrolyzed bioreactor culture with >76% recovery and a final peptide purity of >97%. Antifungal peptide prepared by recombinant and solid phase synthesis methods demonstrated similar activity against Candida sp. in a broth microdilution assay.
authors.affiliation: Xoma (US) LLC, 1545 17th Street, Santa Monica, CA, USA.
authors.forename: P
authors.initials: P
authors.lastname: Gavit
authors.forename: M
authors.initials: M
authors.lastname: Better
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000378:metabolism*; D019149:Bioreactors; D002176:Candida albicans / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
chemical_list: D000935:Antifungal Agents; D011993:Recombinant Fusion Proteins