Liu Wen, Rogge Corina E, Kamensky Yury, Tsai Ah-Lim, Kulmacz Richard J
Department of Internal Medicine, University of Texas Health Science Center at Houston, 6431 Fannin Street, Houston, TX 77030, USA.
Protein Expr Purif. 2007 Dec;56(2):145-52. doi: 10.1016/j.pep.2007.04.010. Epub 2007 Apr 25.
Adrenal cytochrome b561 (cyt b561) is the prototypical member of an emerging family of proteins that are distributed widely in vertebrate, invertebrate and plant tissues. The adrenal cytochrome is an integral membrane protein with two b-type hemes and six predicted transmembrane helices. Adrenal cyt b561 is involved in catecholamine biosynthesis, shuttling reducing equivalents derived from ascorbate. We have developed an Escherichia coli system for expression, solubilization and purification of the adrenal cytochrome. The spectroscopic and redox properties of the purified recombinant protein expressed in this prokaryotic system confirm that the cytochrome retains a native, fully functional form over a wide pH range. Mass spectral analysis shows that the N-terminal signal peptide is intact. The new bacterial expression system for cyt b561 offers a sixfold improvement in yield and other substantial advantages over existing insect and yeast cell systems for producing the recombinant cytochrome for structure-function studies.
肾上腺细胞色素b561（cyt b561）是一个新出现的蛋白质家族的典型成员，该家族广泛分布于脊椎动物、无脊椎动物和植物组织中。肾上腺细胞色素是一种整合膜蛋白，含有两个b型血红素和六个预测的跨膜螺旋。肾上腺cyt b561参与儿茶酚胺的生物合成，穿梭传递来自抗坏血酸的还原当量。我们已经开发了一种大肠杆菌系统，用于肾上腺细胞色素的表达、溶解和纯化。在这个原核系统中表达的纯化重组蛋白的光谱和氧化还原特性证实，该细胞色素在很宽的pH范围内保持天然的、完全功能的形式。质谱分析表明N端信号肽是完整的。用于cyt b561的新细菌表达系统在产量上比现有的用于生产重组细胞色素进行结构功能研究的昆虫和酵母细胞系统提高了六倍，并且还有其他显著优势。
