[Site-specific PEGylation of recombinant lysostaphin]

Abstract

Lysostaphin (Lysn) is an antibacterial metalloendopeptidase that cleaves the pentaglycin bridges in the cell wall of Staphylococci. Although many studies have demonstrated its high activity in vitro, the medical application of Lysn has been hampered by its short half-life in vivo. In order to enhance its stability in vivo without significantly suppressing the enzymatic activity, we designed and tested eight single cysteine substitutions in Lysn for covalent attachment of polyethylene glycol chains (PEGylation). The purified mutants, fully reduced by Dithiothreitol (DTT), were treated with mPEG-MAL(20 kDa). The PEG modification efficiency was above 70% as determined by reverse-phase high-pressure liquid chromatography (HPLC) analysis. The PEG-Lysn proteins were further purified by cation exchange chromatography (MacroCap SP), reaching at least 95% purity. The activities of the PEG-Lysn proteins were determined by the turbidity and minimum inhibitory concentration (MIC) assays. We found that the PEGylated V240C and T244C mutants retained about 50% of the original antibacterial activity of Lysn. Overall, this study will help develop highly stable and active PEG-Lysn to treat systemic S. aureus infections.