Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
Department of Chemistry, University of Florida, 214 Leigh Hall, Gainesville, Florida, 32611, United States of America.
Sci Rep. 2017 Jul 26;7(1):6561. doi: 10.1038/s41598-017-06856-y.
Sortase A (SrtA) is a transpeptidase widely used to site-specifically modify peptides and proteins and shows promise for industrial applications. In this study, a novel strategy was developed for constructing immobilized-SrtA as a robust and recyclable enzyme via direct immobilization of extracellularly expressed SrtA in the fermentation supernatant using magnetic particles. Efficient extracellular SrtA expression was achieved in Escherichia coli through molecular engineering, including manipulation of the protein transport pathway, codon optimization, and co-expression of molecular chaperones to promote expressed SrtA secretion into the medium at high levels. Subsequently, a simple one-step protocol was established for the purification and immobilization of SrtA containing a His-tag from the fermentation supernatant onto a nickel-modified magnetic particle. The immobilized SrtA was proved to retain full enzymatic activity for peptide-to-peptide ligation and protein modification, and was successfully reused for five cycles without obvious activity loss.
Sortase A（SrtA）是一种转肽酶，广泛用于定点修饰肽和蛋白质，并有望在工业应用中得到应用。在这项研究中，开发了一种新的策略，通过使用磁性颗粒直接将发酵上清液中表达的细胞外 SrtA 固定化，将固定化-SrtA 构建为一种坚固且可回收的酶。通过分子工程，包括操纵蛋白质转运途径、密码子优化和共表达分子伴侣，在大肠杆菌中实现了高效的细胞外 SrtA 表达，从而将表达的 SrtA 高水平分泌到培养基中。随后，建立了一种简单的一步法，从发酵上清液中纯化和固定化带有 His 标签的 SrtA 到镍修饰的磁性颗粒上。固定化 SrtA 被证明保留了用于肽到肽连接和蛋白质修饰的完整酶活性，并在没有明显活性损失的情况下成功重复使用了五轮。
