Molecular Cloning and Characterization of a (Lys)6-Tagged Sulfide-Reactive Hemoglobin I from Lucina pectinata.
Molecular Cloning and Characterization of a (Lys)6-Tagged Sulfide-Reactive Hemoglobin I from Lucina pectinata.
复制标题
DOI：
10.1007/s12033-015-9896-8
复制
发表时间：
2015-12
影响因子：
2.6
通讯作者：
López-Garriga J
中科院分区：
文献类型：
--
作者：
Díaz-Ayala R;Moya-Rodríguez A;Pietri R;Cadilla CL;López-Garriga J
将多聚赖氨酸标签与果蝇血红蛋白I(HBI)编码序列融合，采用高效、快速的方法进行纯化。HBI是一种与硫化氢(H_2S)高亲和力结合的血红蛋白，已被用来理解该信号分子的生理相关反应。(Lys)6标记的rHbI在大肠杆菌中表达，并通过阳离子交换基质固定化和大小排斥层析进行纯化。(Lys)6标记的rHbI的结构、结构和功能通过质谱学、X射线散射光谱、光谱和动力学分析进行了鉴定。散射和光谱结果表明，(Lys)6标记的rHbI在结构和功能上与天然蛋白质和(His)6标记的rHbI相似。H_2S的动力学研究表明，缔合(Kon)和解离(Koff)速率常数分别为1.4×10~5/M/S和0.1×10−-3/S。这一结果证实了(Lys)6标记的rHbI以与其同系物相同的高亲和力结合H_2S。
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 (kon) and dissociation (koff) rate constants were 1.4 × 105/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.