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Abstract：
Telethonin anchors the N-terminal region of titin in the Z-disk of the sarcomere by binding to two immunoglobulin-like (Ig) domains (Z1 and Z2) of titin (Z1Z2). Thereby telethonin plays an important role in myofibril assembly and in muscle development and functional regulation. The expression and purification of recombinant telethonin is very challenging. In previous studies, recombinant telethonin expressed from E. coli was refolded in the presence of Z1Z2. Here, we report various strategies to establish a reliable and efficient protocol for the preparation of telethonin and titin Z1Z2 protein. First, a co-expression strategy was designed to obtain soluble Z1Z2/telethonin complexes. The concentration of antibiotics and the type of expression vector were found to be important for achieving high yields of purified complex. Second, the five cysteine residues of telethonin were mutated to serine to avoid severe problems with cysteine oxidation. Third, a short version of telethonin (telethonin1-90) was designed to avoid the proteolytic degradation observed for longer constructs of the protein. The short telethonin formed a highly stable complex with Z1Z2 with no degradation being observed for 30 days at 4 °C. Fourth, an improved refolding protocol was developed to achieve high yields of Z1Z2/telethonin complex. Finally, based on the crystal structure in which Z1Z2 and telethonin1-90 assemble into a 2:1 complex, a single chain fusion protein was designed, comprising two Z1Z2 modules that are connected by flexible linkers N- and C-terminally of the telethonin1-90. Expression of this fusion protein, named ZTZ, affords high yields of soluble expressed and purified protein.
摘要：
Telethonin通过与肌动蛋白（Z1Z2）的两个免疫球蛋白样（Ig）结构域（Z1和Z2）结合，将肌动蛋白的N末端区域锚定在肌节的Z盘中。因此,端黄素在肌原纤维组装以及肌肉发育和功能调节中起重要作用。重组端黄素的表达和纯化是非常具有挑战性的。在以往的研究中,在Z1Z2的存在下,将从大肠杆菌表达的重组的端黄素重折叠。这里,我们报告了各种策略，以建立可靠和有效的方案来制备端黄素和肌动蛋白Z1Z2蛋白。首先,设计了共表达策略以获得可溶性Z1Z2/端黄素复合物。发现抗生素的浓度和表达载体的类型对于获得高产量的纯化复合物是重要的。第二,为了避免半胱氨酸氧化的严重问题,将5个半胱氨酸残基突变为丝氨酸.第三，设计了短版本的端黄素（telethonin1-90），以避免在更长的蛋白质构建体中观察到的蛋白水解降解。短的端黄素与Z1Z2形成高度稳定的复合物，在4℃下30天未观察到降解。第四，开发了一种改进的重折叠方案,以实现Z1Z2/端黄素复合物的高产率.最后,基于Z1Z2和telethonin1-90组装成2:1复合物的晶体结构，设计了一个单链融合蛋白,其包含两个Z1Z2模块,所述模块通过telethonin1-90的N-和C-末端的柔性接头连接。这种融合蛋白的表达，叫ZTZ,提供高产量的可溶性表达和纯化的蛋白质。
