Londer Yuri Y, Pokkuluri P Raj, Orshonsky Valerie, Orshonsky Lisa, Schiffer Marianne
Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA.
Protein Expr Purif. 2006 May;47(1):241-8. doi: 10.1016/j.pep.2005.11.017. Epub 2005 Dec 15.
Multiheme cytochromes c are difficult to produce in heterologous systems. The genome of delta-proteobacterium Geobacter sulfurreducens contains more than a hundred genes coding for c-type cytochromes. Among those are two dodecaheme cytochromes c representing a new class of multiheme cytochromes, whose putative structure is a one-dimensional array of small highly homologous domains that contain three hemes and are covalently bound by short linkers. They are likely to form "nanowires" that are part of the electron transfer chain. We cloned the genes coding for the two cytochromes into a vector we developed for ligation-independent cloning of proteins targeted to the Escherichia coli periplasmic space. We expressed the proteins in E. coli co-transformed with a plasmid harboring the cytochrome c maturation genes. Expression levels were optimized by varying IPTG concentrations used for induction. Although both proteins appeared insoluble or strongly associated with cell membranes, they were solubilized using 0.5 M sodium chloride which was more selective than conventional solubilizing agents, such as HEGA-10 or beta-octylglucoside. The solubilized proteins were dialyzed and purified by cation exchange chromatography followed by gel filtration. Mass-spectrometry analysis confirmed that both purified proteins contained the complete set of covalently attached hemes, 12 per molecule. Their visible spectra were typical of c-type cytochromes. Both proteins were successfully crystallized.
多血红素细胞色素c在异源系统中难以产生。δ-变形菌硫还原地杆菌的基因组包含一百多个编码c型细胞色素的基因。其中有两种十二血红素细胞色素c,它们代表了一类新的多血红素细胞色素,其推测结构是由包含三个血红素且通过短连接子共价结合的小的高度同源结构域组成的一维阵列。它们可能形成作为电子传递链一部分的“纳米线”。我们将编码这两种细胞色素的基因克隆到我们开发的用于将靶向大肠杆菌周质空间的蛋白质进行不依赖连接的克隆的载体中。我们在与携带细胞色素c成熟基因的质粒共转化的大肠杆菌中表达这些蛋白质。通过改变用于诱导的异丙基-β-D-硫代半乳糖苷(IPTG)浓度来优化表达水平。尽管这两种蛋白质似乎不溶或与细胞膜紧密结合,但使用0.5M氯化钠使其溶解,氯化钠比传统的增溶剂(如HEGA-10或β-辛基葡糖苷)更具选择性。将溶解的蛋白质进行透析,然后通过阳离子交换色谱法继以凝胶过滤进行纯化。质谱分析证实两种纯化的蛋白质都含有完整的共价连接的血红素组,每个分子12个。它们的可见光谱是c型细胞色素的典型光谱。两种蛋白质均成功结晶。