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. 2023 Jun;120(6):1694-1701.
doi: 10.1002/bit.28360. Epub 2023 Mar 2.

Engineering active lysostaphin variants that incorporate noncanonical amino acids and characterizing the effects of site-specific PEGylation

Troy Batugal  1 , Geetanjali Pendyala  2 , Luke Tomasovic  2 , Chad Varner  2 , Jeremy D Caplin  3   4 , Alexander M Page  5 , Michelle Davis  5 , Sarah W Satola  5 , Andrés J García  3   4 , Ravi S Kane  2   3
Affiliations

Engineering active lysostaphin variants that incorporate noncanonical amino acids and characterizing the effects of site-specific PEGylation

Troy Batugal et al. Biotechnol Bioeng. 2023 Jun.

Abstract

We describe a facile strategy to identify sites for the incorporation of noncanonical amino acids into lysostaphin-an enzyme that degrades the cell wall of Staphylococcus aureus-while retaining stapholytic activity. We used this strategy to generate active variants of lysostaphin incorporating para-azidophenylalanine. The incorporation of this "reactive handle" enabled the orthogonal site-specific modification of the enzyme variants with polyethylene glycol (PEG) using copper-free click cycloaddition. PEGylated lysostaphin variants could retain their stapholytic activity, with the extent of retention depending on the site of modification and the PEG molecular weight. The site-specific modification of lysostaphin could be useful not only for PEGylation to improve biocompatibility but also for the incorporation of the enzyme into hydrogels and other biomaterials and for studies of protein structure and dynamics. Moreover, the approach described herein could be readily applied to identify suitable sites for the incorporation of reactive handles into other proteins of interest.

Keywords: MRSA; antibiotic-resistant bacteria; lysins; lysostaphin; noncanonical amino acid.

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References

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