In Vitro and In Planta Cyclization of Target Peptides Using an Asparaginyl Endopeptidase from Oldenlandia affinis

Cyclization of the peptide backbone by connecting the N- and C-terminus can endow target peptides with favorable properties, such as increased stability or potential oral bioavailability. However, there are few tools available for carrying out this modification. Asparaginyl endopeptidases (AEPs) are a class of enzymes that typically work as proteases, but a subset is highly efficient at cyclization of the peptide backbone. In this chapter we describe how to utilize a cyclizing AEP (OaAEP1_b) to produce backbone-cyclized peptides both in planta and in vitro. Using the in planta method, OaAEP1_b and the target precursor peptide are coexpressed in the leaves of the model plant Nicotiana benthamiana, and cyclization of the target peptide occurs in planta. Using the in vitro method, purified recombinant OaAEP1_b produced in bacteria is used to cyclize the target precursor peptide in vitro.