title: A chromatography-focused bioprocess that eliminates soluble aggregation for bioactive production of a new antimicrobial peptide candidate.
abstract: Human beta defensins (hBDs) are an important class of antimicrobial peptides (AMPs), which provide the host with innate protection from bacteria, fungi and viruses. Human β-defensin-25 (hBD25) is a new hBD variant which has been recently discovered in the male genital tract. Since its discovery, hBD25 was hypothesized to play a key role in protection against genital tract infection, which has significantly increased mortality rates in the last decade. However, further studies to confirm the role of hBD25 are hindered by the lack of sufficient amounts of pure hBD25 for clinical studies. This study reports the first successful development of an efficient and low cost chromatography-oriented bioprocess for production of hBD25. hBD25 was expressed predominantly as soluble aggregates although the peptide was co-expressed with a Maltose Binding Protein (MBP) fusion tag in E. coli. The soluble aggregates were disrupted by denaturation-reduction of the hBD25, followed by an in vitro size exclusion chromatography refolding step which readily yielded bioactive and purified hBD25 peptides at 90% purity. The refolded hBD25 showed antimicrobial activity against E. coli K12 at a minimal inhibitory concentration of 60 μg/mL. With an overall hBD25 bioprocess yield of 48% obtained, this bioprocess will open the way for detailed clinical studies of hBD25, and serve as a generic platform for efficient recovery of other 'fusion protein'-derived peptides that inevitably exist as soluble aggregates.
authors.affiliation: School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore.
authors.forename: Xiang
authors.initials: X
authors.lastname: Li
authors.forename: Susanna Su Jan
authors.initials: SS
authors.lastname: Leong
mesh_terms: D000900:Anti-Bacterial Agents / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D002850:Chromatography, Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008826:Microbial Sensitivity Tests; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D012995:Solubility; D023083:beta-Defensins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins