来自栉孔扇贝的（Lys）6标记的硫化物反应性血红蛋白I的分子克隆与表征
Molecular Cloning and Characterization of a (Lys)6-Tagged Sulfide-Reactive Hemoglobin I from Lucina pectinata.
作者信息
Díaz-Ayala Ramonita, Moya-Rodríguez Andrés, Pietri Ruth, Cadilla Carmen L, López-Garriga Juan
出版信息
Mol Biotechnol. 2015 Dec;57(11-12):1050-62. doi: 10.1007/s12033-015-9896-8.
A poly-Lys tag was fused to the Lucina pectinata hemoglobin I (HbI) coding sequence and purified using an efficient and fast process. HbI is a hemeprotein that binds hydrogen sulfide (H2S) with high affinity and it has been used to understand physiologically relevant reactions of this signaling molecule. The (Lys)6-tagged rHbI construct was expressed in E. coli and purified by immobilization on a cation exchange matrix, followed by size-exclusion chromatography. The identity, structure, and function of the (Lys)6-tagged rHbI were assessed by mass spectrometry, small and wide X-ray scattering, optical spectroscopy, and kinetic analysis. The scattering and spectroscopic results showed that the (Lys)6-tagged rHbI is structurally and functionally analogous to the native protein as well as to the (His)6-tagged rHbI. Kinetics studies with H2S indicated that the association (k on) and dissociation (k off) rate constants were 1.4 × 10(5)/M/s and 0.1 × 10(-3)/s, respectively. This results confirmed that the (Lys)6-tagged rHbI binds H2S with the same high affinity as its homologue.
将一个聚赖氨酸标签融合到紫扇贝血红蛋白I（HbI）的编码序列上，并通过高效快速的方法进行纯化。HbI是一种血红素蛋白，能与硫化氢（H₂S）高亲和力结合，已被用于了解这种信号分子的生理相关反应。带有（Lys）₆标签的重组HbI构建体在大肠杆菌中表达，并通过固定在阳离子交换基质上，随后进行尺寸排阻色谱法进行纯化。通过质谱、小角和广角X射线散射、光谱学以及动力学分析对带有（Lys）₆标签的重组HbI的身份、结构和功能进行了评估。散射和光谱学结果表明，带有（Lys）₆标签的重组HbI在结构和功能上与天然蛋白以及带有（His）₆标签的重组HbI相似。对H₂S的动力学研究表明，结合（k on）和解离（k off）速率常数分别为1.4×10⁵/M/s和0.1×10⁻³/s。这一结果证实，带有（Lys）₆标签的重组HbI与硫化氢结合的亲和力与其同源物相同。
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