Harris Karen S, Poon Simon, Quimbar Pedro, Anderson Marilyn A
Hexima Limited, Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia.
Methods Mol Biol. 2019;2012:211-235. doi: 10.1007/978-1-4939-9546-2_12.
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) to produce backbone-cyclized peptides both in planta and in vitro. Using the in planta method, OaAEP1 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 produced in bacteria is used to cyclize the target precursor peptide in vitro.
通过连接N端和C端使肽主链环化，可以赋予目标肽良好的性质，如提高稳定性或潜在的口服生物利用度。然而，用于进行这种修饰的工具很少。天冬酰胺内肽酶（AEPs）是一类通常作为蛋白酶起作用的酶，但其中一部分在肽主链环化方面效率很高。在本章中，我们描述了如何利用一种环化AEP（OaAEP1）在植物体内和体外产生主链环化的肽。使用植物体内方法，OaAEP1和目标前体肽在模式植物本氏烟草的叶片中共表达，目标肽在植物体内发生环化。使用体外方法，细菌中产生的纯化重组OaAEP1用于在体外使目标前体肽环化。
