Click Chemistry for Site Directed Labeling of Surface Displayed Proteins on Escherichia coli

Nienberg C, Retterath A, Jose J

Abstract

The Autodisplay technology is based on the natural secretion mechanism of autotransporter proteins on the surface of bacterial cells [1]. The surface display of human protein kinase CK2 on Escherichia colihas previously been shown [2]. Combining this technique with the incorporation of an unnatural amino acid into proteins facilitates bioorthogonal reactions and expands the capabilities of protein chemistry.This study reports the successful incorporation of an unnatural amino acid into the surface displayed fusion protein CK2β followed by a bioorthogonal click reaction. CK2β was used as a model protein and isthe regulatory subunit of the human protein kinase CK2. The DNA triplet encoding Tyr108 of CK2β was chosen and mutated to the amber DNA stop codon, TAG. By suppression of the mutation with an ambersuppressor tRNA, the unnatural amino acid para acidophenylalanine (pAzF) [3] could be incorporated at this position. Performing the SPAAC click reaction (Strain-Promoted Alkyne-Azide Cycloaddition) [4]using a dibenzylcyclooctyne-fluorophore (DBCO fluorophore) resulted in a side specific labeling of CK2β fusion protein on the surface of E. coli, which was evaluated by flow cytometry and SDS-PAGE. Thefunctionality of the obtained regulatory CK2β subunit activating the catalytically active CK2α subunit was confirmed by capillary electrophoresis. This innovative procedure of labeling proteins on the outermembrane of bacterial cells could be a significant advancement for screening assays or protein interaction studies with whole cells, cell lysates or isolated outer membrane proteins.