关键词: 蛋白质合成, 蛋白质定点修饰, Sortase A转肽酶, Butelase 1转肽酶, Subtilisin人工连接酶

Abstract:

As a hot-spot of synthetic biology, chemical protein synthesis and modification have been widely applied to generate and functionalize naturally inaccessible proteins to meet scientific or pharmaceutical demands. Breaking away from the restrictions on the amino acids utilized in ribosomal protein synthesis, protein synthesis, and modification through the ligation of synthetic peptides have provided a platform for preparing the unnatural proteins that contain hundreds of residues, which realizes the artificial design of protein on the atomic scale. Although several chemical ligation methods have been reported, they are confronted with the defects like limited junction choices and complicated substrate preparations, impelling the scientists to find out alternative solutions and develop enzymatic strategies fit for different applications. As a group of popular peptide ligation methods, enzymatic ligation strategies not only expand our understanding of protein, but also exhibit great advantages in the industrial production of pharmaceutical peptides.Sortase A (SrtA) has been extensively utilized for protein terminal modification. The researchers from various fields have attempted to develop novel applications based on SrtA, for instance, characterizing the surface proteins of eukaryotic cells and expanding the choices of phage capsid proteins. Butelase 1 from a cyclopeptide-producing plant (Clitoria ternatea) is a highly efficient peptide ligase for peptide cyclization. Large proteins like GFP could be cyclized with excellent yield (>95%) as well, and Butelase 1 can be used for synthesizing peptide dendrimers, preparing protein thioesters, and modifying bacterial surfaces as well. Subtilisin-derived ligase is engineered from subtilisin and thought as a powerful tool for peptide cyclization, protein synthesis and terminal modification. In recent years, based on a calcium-independent subtilisin variant, a thermostable and organic solvent-tolerant peptide ligase was constructed and termed Peptiligase, which was further engineered afterward. Omniligase-1 is a universal ligase bearing broad sequence compatibility, while Thymoligase is specially designed for the production of the pharmaceutic peptide Thymosin-α1. These Peptiligase variants exhibit great advantages for industrial applications and have been considered as the most impressive enzymatic approach to date. The development of the existing enzymatic methods is anticipated to be ameliorated through enzyme engineering, to which rational design and directed evolution have contributed a lot. At the moment of computational protein design communicating with chemical protein synthesis, we anticipate that more peptide ligases would be discovered or designed. They may serve for generations of previously hard-to-access modified proteins.

Key words: protein synthesis, protein modification, Sortase A, Butelase 1, Subtilisin-derived ligases