Goncharuk M V, Shul'ga A A, Ermoliuk Ia S, Tkach E N, Goncharuk S A, Pustovalova Iu E, Mineev K S, Bocharov É V, Maslennikov I V, Arsen'ev A S, Kirpichnikov M P
Mol Biol (Mosk). 2011 Sep-Oct;45(5):892-902.
A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.
表皮生长因子受体家族（ErbB）是一类重要的受体酪氨酸激酶，在细胞生长、发育和分化过程中起主导作用。这些受体的跨膜结构域通过侧向同源和异源二聚化在质膜上转导生化信号。ErbB跨膜结构域与去污剂或脂质形成的复合物尺寸相对较小，这使得人们能够使用三维异核高分辨率核磁共振光谱来研究其详细的空间结构。在此，我们描述了一种有效的表达系统和纯化程序，用于从ErbB家族的四个代表成员ErbB1、ErbB2、ErbB3、ErbB4制备跨膜肽，以进行结构研究。重组肽在大肠杆菌BL21(DE3)pLysS中作为硫氧还蛋白A的C端延伸片段产生。融合蛋白的切割用人肠激酶轻链完成。通过一种简单便捷的程序，即依次进行固定金属亲和色谱和阳离子交换色谱步骤，分离得到了几（10 - 30）毫克纯化的同位素标记跨膜肽。将纯化后的肽在脂质/去污剂环境（胶束或双分子层）中重构，并使用动态光散射、圆二色光谱和核磁共振光谱进行表征。所得数据表明，纯化后的ErbB跨膜肽适用于使用高分辨率核磁共振光谱对其同源和异源二聚体复合物进行结构和动力学研究。
