Preparing Active Recombinant AEP free of Acid-activation.

Abstract Asparaginyl endopeptidase is a useful biocatalyst for site-specific protein bioconjugation. However, existing recombinant protocol is lengthy requiring cap removal of the zymogenic enzymes through acid activation and extra chromatographic steps. Here, we describe an activation-free approach for AEP preparation where the cap and core domains are prepared as separate entities during gene expression. An AEP variant from Oldenlandia affinis (OaAEP1b-C247A) can be prepared without the need of acid treatment giving a yield of ~1.5-2.2 mg of enzyme per litre of culture. Likely because of a decrease in chromatography time and an increase of homogeneity, activity of the split AEP was found to be ~3-fold higher than those obtained using existing protocols, highlighting its potential usefulness.