Approaches that allow site-specific and bioorthogonal chemical modification of proteins are highly desirable for protein engineering applications. A novel approach to incorporate bioorthogonal reactive handles for site-specific protein modification entails the use of ligase-mediated post-translational modifications. This approach specifically uses the enzyme lipoic acid ligase (LplA) to modify proteins, which contain a unique peptide tag (LAP), with clickable azide groups that may then serve as sites for polymer attachment or immobilization. In exploring the potential utility of the LAP/LplA system, we have demonstrated that the LAP sequence can be recognized and ligated by LplA when genetically inserted at various, internal positions within the primary sequence of the model protein, GFP. Ligation at these positions, which correspond to unique locations within the protein structure, proceeded efficiently with greater than 90 percent conversion observed after one hour for each of the insertion sites. Further, strong expression levels were obtained for GFP constructs with single as well as double, simultaneous LAP insertions. In illustrating the potential of internal LAP insertion sites, azide containing GFP molecules were site specifically pegylated with 5 kDa alkyne functionalized polyethylene glycol. These reactions also achieved high conversions, generating molecular populations with a greater degree of homology than those observed through pegylation with non-specific chemistries. Also, by combining an internal insertion site with an N-terminal LAP sequence, strong and uniform surface coverage was able to be achieved by pegylation. These results demonstrate the potential of the LAP/LplA system for introducing highly specific post-translational modification sites at designed locations within a protein structure.

Ligase-mediated bioorthogonal insertion of click reactive groups for site-specific protein modification

249th National Meeting (2015)

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