Peptidoglycan-Targeting Staphylolytic Enzyme Lysostaphin as a Novel and Efficient Protease toward Glycine-Rich Flexible Peptide Linkers

Liu Z. Gong G. Li Y. Xu Q. Akimbekov N. Zha J. Wu X.
5 April 2023 American Chemical Society

Journal of Agricultural and Food Chemistry
2023 #71 Issue 13 5293 - 5301 pp.

Glycine-rich flexible peptide linkers have been widely adopted in fusion protein engineering; however, they can hardly be cleaved for the separation of fusion partners unless specific protease recognition sites are introduced. Herein, we report the use of the peptidoglycan-targeting staphylolytic enzyme lysostaphin to directly digest the glycine-rich flexible linkers of various lengths including oligoglycine linkers and (G₄S)_x linkers, without the incorporation of extra amino acids. Using His-MBP-linker-LbCpf1 as a model substrate, we show that both types of linkers could be digested by lysostaphin, and the digestion efficiency improved with increasing linker length. The enzyme LbCpf1 retained full activity after tag removal. We further demonstrated that the proteolytic activity of lysostaphin could be well maintained under different environmental conditions and in the presence of a series of chemical reagents at various concentrations that are frequently used in protein purification and stabilization. In addition, such a digestion strategy could also be applied to remove the SUMO domain linked to LwCas13a via an octaglycine linker. This study extends the applications of lysostaphin beyond an antimicrobial reagent and demonstrates its potential as a novel, efficient, and robust protease for protein engineering.

flexible peptide linker , fusion protein , glycine-rich , lysostaphin , protease

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School of Food and Biological Engineering, Shaanxi University of Science and Technology, Shaanxi, Xi’an, 710021, China
Department of Biotechnology, Al-Farabi Kazakh National University, Almaty, 050040, Kazakhstan

School of Food and Biological Engineering
Department of Biotechnology

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