Turnay J, Pfannmüller E, Lizarbe M A, Bertling W M, von der Mark K
Max-Planck Society, Medical Clinic III, University of Erlangen-Nürnberg, Germany.
J Cell Biochem. 1995 Jun;58(2):208-20. doi: 10.1002/jcb.240580210.
We have cloned the full coding cDNA sequence of chicken annexin V and of a mutant lacking 8 amino acid residues of the N-terminal tail for prokaryotic expression. Both proteins were synthesized in Escherichia coli upon induction with isopropyl thio-beta-D-galactoside, and were purified following two different protocols: one based on the ability of these proteins to interact reversibly with liposomes in the presence of calcium, and the other based on two sequential ion-exchange chromatographic steps. Spectroscopical analysis of recombinant annexin V revealed that binding of calcium did not change the circular dichroism spectra indicating no significant changes on the secondary structure; however, a conformational change affecting the exposition to the solvent of the tryptophan residue 187 was detected by analysis of fluorescence emission spectra. Recombinant annexin V binds with high affinity to collagen types II and X, and with lower affinity to collagen type I in a calcium-independent manner. Heat denaturing of collagen decreases this interaction while pepsin-treatment of collagen almost completely abolishes annexin V binding. Mutated annexin V interacts with collagen in a similar way as the nonmutated recombinant protein, indicating that the N-terminal tail of annexin V is not essential for collagen binding.
我们克隆了鸡膜联蛋白V以及N端尾部缺失8个氨基酸残基的突变体的完整编码cDNA序列，用于原核表达。两种蛋白经异丙基硫代-β-D-半乳糖苷诱导后在大肠杆菌中合成，并通过两种不同的方法进行纯化：一种基于这些蛋白在钙存在下与脂质体可逆相互作用的能力，另一种基于两个连续的离子交换色谱步骤。重组膜联蛋白V的光谱分析表明，钙的结合并未改变圆二色光谱，这表明二级结构没有显著变化；然而，通过荧光发射光谱分析检测到一种构象变化，影响了色氨酸残基187与溶剂的接触。重组膜联蛋白V以钙非依赖的方式与II型和X型胶原高亲和力结合，与I型胶原的亲和力较低。胶原的热变性降低了这种相互作用，而用胃蛋白酶处理胶原几乎完全消除了膜联蛋白V的结合。突变的膜联蛋白V与胶原的相互作用方式与未突变的重组蛋白相似，这表明膜联蛋白V的N端尾部对于胶原结合并非必需。
