The site-specific chemical modification of proteins through incorporation of noncanonical amino acids enables diverse applications, such as imaging, probing, and expanding protein functions, as well as to precisely engineer therapeutics. Here we report a general strategy that allows the incorporation of noncanonical amino acids into target proteins using the amber suppression method and their efficient secretion in the biotechnological relevant expression host Bacillus subtilis. This facilitates efficient purification of target proteins directly from the supernatant, followed by their functionalization using click chemistry. We used this strategy to site-specifically introduce norbornene lysine into a single chain antibody and functionalize it with fluorophores for the detection of human target proteins.

Center for Integrated Protein Science at the Department of Chemistry Ludwig Maximilians University Munich Butenandtstraße 5 13 Munich 81377 Germany

Institute of Organic Chemistry and Biochemistry of the CAS Flemingovo nám 2 Prague 6 CZ 166 10 Czech Republic

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