Genetic code expansion, protein expression and protein functionalisation in Bacillus subtilis

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 high potential microbial production system Bacillus subtilis. This facilitates efficient purification of target proteins directly from the supernatant, followed by their functionalisation using click chemistry. We used this strategy to introduce norbornene lysine site-specifically into a single chain antibody and functionalise it with fluorophores for the detection of human target proteins.