Multiple linkages via sortase-mediated ligation with the example of protein-polymer conjugates

分选酶是存在于革兰氏阳性细菌的细胞壁中的酶，其将表面蛋白共价连接到细胞壁。分选酶A在肽基序LPXTG和寡聚甘氨酸之间形成肽键。这种连接技术被称为分选酶介导的连接（SML）或分选标记，并发展成为基础研究中常用的工具。使用分选酶A，可以将蛋白质连接到另一个生物分子、小合成分子、聚合物或表面。因此，底物仅需要配备有C-末端LPXTG和N-末端Gx序列。SML的一个弱点是形成的键（LPXTGGG）再次代表酶的底物。该反应是可逆的，因此，形成的键可以被酶再次裂解。如今，几种可能性允许改变反应的平衡，使得能够以几乎定量的产率进行连接。然而，通过一种分选酶形成连续键仍然是不可能的，或者只能通过苛刻的策略来实现。因此，蛋白质链、表面上的多层蛋白质以及在C-和N-末端修饰的蛋白质难以接近。在这个项目的过程中，应该开发一种有效的方法，通过分选酶A连接几个构件。这应该用蛋白质-聚合物缀合物的例子来说明，并且可以获得具有连接到蛋白质的两个不同聚合物链的缀合物类型。此外，蛋白质寡聚体的生成过程应类似于梅里菲尔德肽合成。该项目包括用于连接蛋白质C-和N-末端的聚合物嵌段的合成。在此基础上，利用SML合成蛋白质-聚合物复合物。尚未显示使用分选酶A的连续连接，其中所形成的键不能被酶切割。这应该通过利用形成不再是分选酶A的底物的β-发夹结构的氨基酸序列来实现。该方法将首先用蛋白质的多个（不受控制的）连接来证明。为了确保三个或更多个结构单元的受控连接，首先必须封闭一个蛋白质末端。该程序将使用掩蔽的低聚甘氨酸开发，并通过连接几个蛋白质块来显示。随后，该策略应转移到两个聚合物链连接到一个蛋白质。

Sortases are enzymes occurring in the cell wall of gram-positive bacteria that covalently link surface proteins to the cell wall. Sortase A forms a peptide bond between the peptide motif LPXTG and an oligoglycine. This ligation technique is known as sortase-mediated ligation (SML) or sortagging and developed to a frequently used tool in basic research. Using sortase A, a protein can be ligated to another biomolecule, a small synthetic molecule, a polymer or a surface. Therefore, the substrates only need to be equipped with a C-terminal LPXTG and an N-terminal Gx sequence. A weakness of SML is that a formed bond (LPXTGGG) represents again a substrate for the enzyme. The reaction is reversible and, consequently, a formed bond can be cleaved again by the enzyme. Nowadays, several possibilities allow to shift the equilibrium of the reaction enabling ligations in nearly quantitative yield. However, the formation of consecutive bonds by one sortase is still impossible or can be only reached via demanding strategies. Therefore, chains of proteins, multiple layers of proteins on surfaces, and proteins modified at both C- and N-termini are difficult to access. In the course of this project, an efficient approach should be developed to link several building blocks by means of sortase A. This should be shown with the example of protein-polymer conjugates and give access to a type of conjugates with two different polymer chains linked to a protein. Furthermore, protein oligomers should be generated in a procedure similar to the Merrifield peptide synthesis.The project consists of the synthesis of polymer blocks for the ligation to both C- and N-terminus of a protein. On this basis, protein-polymer conjugates will be synthesized by SML. Consecutive ligations using sortase A, where the formed bonds could not be cleaved by the enzyme, have not been shown yet. This should be reached by utilizing amino acid sequences that form β-hairpin structures which are not a substrate for sortase A anymore. The approach will be demonstrated first with the multiple (uncontrolled) ligation of a protein. To ensure a controlled ligation of three or more building blocks, one protein terminus has to be blocked at first. This procedure will be developed using masked oligoglycines and shown with the ligation of several protein blocks. Subsequently, the strategy should be transferred to the linkage of two polymer chains to one protein.