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Secretory ranalexin produced in recombinant Pichia pastoris exhibits additive or synergistic bactericidal activity when used in combination with polymyxin B or linezolid against multi‐drug resistant bacteria
Author(s) -
Aleinein Rasha Abou,
Schäfer Holger,
Wink Michael
Publication year - 2014
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201300282
Subject(s) - microbiology and biotechnology , pichia pastoris , escherichia coli , antibiotics , linezolid , polymyxin b , biology , antimicrobial , staphylococcus aureus , bacteria , antimicrobial peptides , polymyxin , peptide , recombinant dna , biochemistry , vancomycin , gene , genetics
Ranalexin, a cationic peptide from frogs, is a potent therapeutic antimicrobial peptide (AMP). Its limited availability is an obstacle for a wider application. A high‐level production of AMPs via bioengineering is possible but remains a challenging task. In the current study, we investigated the potential antibacterial properties of recombinant ranalexin, expressed in the yeast Pichia pastoris. A 78‐bp DNA fragment encoding the mature ranalexin peptide with a 6‐His tag on its C‐terminus was designed using the preferred codon usage of P. pastoris . The gene was inserted into pPICZaA and transformed into competent cells of P. pastoris strain KM71. The yield of secretory ranalexin reached up to ∼6 mg/L culture. Time‐kill curve analysis of ranalexin against both Escherichia coli and methicillin‐resistant Staphylococcus aureus (MRSA) demonstrated a concentration‐dependent rapid bactericidal activity. In checkerboard assays, the combinations of ranalexin with the established antibiotics polymyxin B or linezolid reduced the MIC additively in most tested bacteria. Time‐kill assays indicated a significant synergism in E. coli and MRSA when ranalexin was used in combination with antibiotics, even at concentrations of 1/4 MIC or 1/2 MIC of ranalexin, respectively. Thus we propose that secretory ranalexin produced in P. pastoris could be a useful tool to unravel ranalexin's biological function and for use in future in vivo studies against multi‐resistant bacterial infections.