Site-directed modification of genetically engineered Proteus sp. lipase K107 variants with a polyethylene glycol derivative

• We report a novel method for site-directed PEGylation of protein.
• Linear mPEGs of various sizes were functionalized via dopamine.
• PEG derivates were used for site-specific PEGylation of enzyme with a single Cys residue.
• The PEGylated enzymes maintained their secondary structures and activities, which was higher in low molecular mass PEG conjugates.
• Conjugates with the site of polymer coupling near the catalytic centre were more stable.

The covalent attachment of PEG to target proteins, known as PEGylation, has broad biomedical and biotechnological applications. Particularly, site-specific PEGylation is widely used owing to its unique properties of preserving bioactivity, improving the stability of conjugated proteins, and achieving a high degree of homogeneity. In this work, linear mPEGs of various sizes (MW = 5, 12, and 20 kDa) were functionalized via dopamine and used for site-specific PEGylation of Proteus sp. lipase K107 derivatives with a single Cys residue introduced by site-directed mutagenesis on the solvent-accessible surface of the protein. The specificity of conjugation was verified by SDS-PAGE and MALDI-TOF mass spectrometry, and the secondary structures of the conjugates were verified by circular dichroism. PEGylated enzymes retained their activity, which was higher in low molecular mass PEG conjugates. Importantly, both pH and thermal stability of enzymes were enhanced by PEGylation, especially at basic pH and above room temperature. Moreover, conjugates with the site of polymer coupling near the catalytic centre were more stable. These results demonstrate a novel, efficient method of site-specific protein modification via catechol-functionalized PEG that could potentially be applied to other enzymes.

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