Zheng Kai, Makagiansar Irwan T, Wang Mengmeng, Urbauer Jeffrey L, Kuczera Krzysztof, Siahaan Teruna J
Department of Molecular Biosciences, The University of Kansas, Lawrence, KS 66045, USA.
Protein Expr Purif. 2004 Jan;33(1):72-9. doi: 10.1016/j.pep.2003.08.021.
The objective of this work was to produce unlabeled and 15N-labeled EC4 domain protein from E-cadherin for studying its structure and binding properties to other EC domains as well as to E-cadherin peptides. The EC4 domain of E-cadherin was expressed in Escherichia coli from the vector pASK-IBA6 and localized in the periplasmic space of E. coli. This protein contains a Streptag sequence at the N-terminus, and thus was purified using a Strep-Tactin affinity column. However, at high concentrations the 15N-labeled EC4 protein showed an unstable conformation. Conditions for stabilizing the conformation of this protein were evaluated using CD spectroscopy. The CD results showed that this protein has high conformational stability in Tris buffer at pH 6.0 in the presence of 10 mM calcium chloride.
这项工作的目的是制备来自E-钙黏蛋白的未标记和15N标记的EC4结构域蛋白，以研究其结构以及与其他EC结构域和E-钙黏蛋白肽的结合特性。E-钙黏蛋白的EC4结构域在大肠杆菌中从载体pASK-IBA6表达，并定位于大肠杆菌的周质空间。该蛋白在N端含有一个链霉亲和肽序列，因此使用链霉亲和素亲和柱进行纯化。然而，在高浓度下，15N标记的EC4蛋白显示出不稳定的构象。使用圆二色光谱法评估稳定该蛋白构象的条件。圆二色光谱结果表明，在存在10 mM氯化钙的情况下，该蛋白在pH 6.0的Tris缓冲液中具有高构象稳定性。
