Improving the use of asparaginyl endopeptidase for biocatalytic applications

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The formation of peptidyl amide linkages is a fundamental reaction in biology yet it is an exceptionally important reaction used in both chemical and synthetic biology research. It is the most commonly employed reaction for peptide ligation and backbone cyclization. Chemically modified proteins have enabled novel approaches towards in vivo imaging, diagnostics and other applied biotechnological research. Furthermore, bio-conjugation, ligation and cyclization of peptides have led to advances in chemical biology for the generation of drug derivatives. Protein and peptidyl drug derivatives may enable access to drug targets that are currently "undruggable". Therefore, an efficient method of peptide ligation and cyclization would be highly desirable.Naturally occurring enzymes with protein ligase activities are relatively rare. It was suggested that asparaginyl endopeptidases (AEP) facilitate peptide backbone macro-cyclisation in certain cyclotide producing plant families. Indeed, OaAEP1 was isolated from a cyclotide producing plant, Oldenlandia affinis, and recombinantly expressed from E. coli. Structural and mutation studies indicated that the enzyme was a cysteine protease with a relatively short recognition sequence (three amino acid residues, C-terminal NGL). Furthermore, a mutant with high catalytic efficiency was discovered. Thus highlighting the potential of OaAEP1 as a biocatalyst for peptide cyclization.This investigation proposes to further develop OaAEP1 as an efficient biocatalyst for peptide cyclization. Initially, it was of interest to further validate the application and the substrate scope of OaAEP1. Subsequently, the investigation focuses on the direct expression of active OaAEP1 in E. coli. The protein is currently expressed in E. coli as a zymogen, which requires activation in low pH conditions. Direct expression of the active enzyme results in detrimental effects on cell viability. Strategies such as encapsulation and protein trafficking were explored in an attempt to conceal the toxicity of the active protein. Together, this work will enable the use of OaAEP1 as a versatile tool used in chemical and synthetic biology research.