重组基因表达和1 h NMR特征kringle (2 + 3) supermodule:光谱/功能个性的纤溶酶原kringle物领域。
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胡Sohndel年代,CK,马蒂D, Affolter M,夏勒J, Llinas M, Rickli EE
重组基因表达和1 h NMR特征kringle (2 + 3) supermodule:光谱/功能个性的纤溶酶原kringle物领域。
生物化学。1996年2月20日,35 (7):2357 - 64。
- PubMed ID
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8652577 (在PubMed]
- 文摘
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纤溶酶原kringle物2 (K2HPg)和kringle 3 (K3HPg)模块发生在串联的多肽片段内提供血纤维蛋白溶酶的重链。K2HPg和K3HPg纤溶酶原kringle物领域中是独一无二的,他们也都与对方通过Cys169-Cys297 (Cys4的K2HPg Cys43 K3HPg, kringle编号惯例)二硫桥,从而生成一个K2HPg-K3HPg“supermodule”。kringle(2 + 3)序列的人类plasminogen (r-EE [K2HPgK3HPg] DS)在大肠杆菌表达,使用一个包含噬菌体T5启动子的表达载体/运营商N250PSN250P29和氨基端hexahistidine的基码标签,以确保隔离重组蛋白的亲和色谱法对镍(2 +)-nitrilotriacetic酸变性、减少条件下/琼脂糖。Kringle(2 + 3)复合的谷胱甘肽氧化还原缓冲区。通过利用kringle 2,赖氨酸亲和力的蛋白质纯化了lysine-Bio-Gel亲和色谱法。重组kringle(2 + 3)是由氨基酸组成,n端序列和质量的决心。1 h NMR谱表明,完整r-K2HPgK3HPg正确折叠。通过参考光谱的个人kringles r-K2HPg r-K3HPg,共振K2HPg和K3HPg组件的完整的频谱r-K2HPgK3HPg很容易区分。严格守恒Leu46残渣(kringle残渣号公约)收益率delta-methyl K2HPg和K3HPg信号特点,表现出-0.87和-0.94 ppm的化学变化,分别是有别于K1HPg, K4HPg, K5HPg, (-1.04 - -1.05 ppm)。因此,轨迹Leu46信号从K2HPg和K3HPg解决其他kringles,可以确定明确的光谱K1HPgK2HPgK3HPg elastolytic片段Glu-plasminogen的纤溶酶原和光谱物。 Overall, r-K2HPgK3HPg exhibits broader resonance line widths than does the K1HPg component, consistent with a lesser mobility of the K2HPgK3HPg segment within the K1HPgK2HPgK3HPg fragment, a reflection of the extra structural constraint imposed by the disulfide bridge linking K2HPg to K3HPg. The ligand 6-aminohexanoic acid (6-AHA), which is known to interact with r-K2HPg but not with r-K3HPg, selectively perturbs K2 aromatic signals in the intact r-K2HPgK3HPg spectrum while leaving K3 resonances largely unaffected. Association constant (K(a)) values for 6-AHA determined from 1H NMR ligand titration experiments yield K(a) approximately 2.2 +/- 0.3 mM(-1) for the intact r-K2HPgK3HPg, comparable to K(a) approximately 2.3 +/- 0.2 mM(-1) determined for the isolated r-K2HPg, which demonstrates that the interactions of 6-AHA with the K2HPg ligand-binding site are not significantly affected by the neighboring K3HPg domain within the intact r-K2HPgK3HPg supermodule.