Improving the use of asparaginyl endopeptidase for biocatalytic applications

肽基酰胺键的形成是生物学中的基本反应，但它是化学和合成生物学研究中使用的非常重要的反应。它是肽连接和骨架环化最常用的反应。化学修饰的蛋白质使体内成像、诊断和其他应用生物技术研究成为可能。此外，肽的生物缀合、连接和环化已经导致用于产生药物衍生物的化学生物学的进步。蛋白质和肽基药物衍生物可能能够获得目前“不可用药”的药物靶点。因此，非常需要一种有效的肽连接和环化方法。有人建议，天冬酰胺酰内肽酶（AEP）促进某些环肽生产植物家族的肽骨架大环化。事实上，OaAEP 1是从产生环肽的植物Oldenlandia affinis中分离的，并从E.杆菌结构和突变研究表明，该酶是一种半胱氨酸蛋白酶，具有相对较短的识别序列（三个氨基酸残基，C-末端NGL）。此外，还发现了一个具有高催化效率的突变体。因此，突出了OaAEP 1作为肽环化的生物催化剂的潜力。本研究建议进一步开发OaAEP 1作为一个有效的生物催化剂的肽环化。最初，进一步验证OaAEP 1的应用和底物范围是有意义的。随后，研究了活性OaAEP 1在大肠杆菌中的直接表达。杆菌该蛋白目前在E.大肠杆菌作为酶原，其需要在低pH条件下活化。活性酶的直接表达导致对细胞活力的有害影响。探索了诸如包封和蛋白质运输的策略，试图隐藏活性蛋白的毒性。总之，这项工作将使OaAEP 1成为化学和合成生物学研究中的通用工具。

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.