Price N J, Brennan L, Faria T Q, Vijgenboom E, Canters G W, Turner D L, Santos H
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal.
Protein Expr Purif. 2000 Dec;20(3):444-50. doi: 10.1006/prep.2000.1318.
Heterologous expression of c-type cytochromes in the periplasm of Escherichia coli often results in low soluble product yield, apoprotein formation, or protein degradation. We have expressed cytochrome c from Methylophilus methylotrophus in E. coli by coexpression of the gene encoding the cytochrome (cycA) with the host-specific cytochrome c maturation elements, within the ccmA-H gene cluster. Aerobic cultures produced up to 10 mg holoprotein per liter after induction with IPTG. In the absence of the maturation factors E. coli failed to produce a stable haem protein. Cytochrome c" isolated from the natural host was compared with the recombinant protein. No structural differences were detected using SDS-PAGE, UV-Visible spectroscopy, differential scanning calorimetry, and (1)H-NMR spectroscopy. The success in expressing the mature cytochrome c in E. coli allows the engineering of the cycA gene by site-directed mutagenesis thereby providing an ideal method for producing mutant protein for studying the structure/function relationship.
c型细胞色素在大肠杆菌周质中的异源表达常常导致可溶性产物产量低、脱辅基蛋白形成或蛋白质降解。我们通过在ccmA-H基因簇内将编码细胞色素（cycA）的基因与宿主特异性细胞色素c成熟元件共表达，在大肠杆菌中表达了嗜甲基甲基ophilus的细胞色素c。用IPTG诱导后，好氧培养物每升可产生高达10毫克的全蛋白。在没有成熟因子的情况下，大肠杆菌无法产生稳定的血红素蛋白。将从天然宿主中分离的细胞色素c"与重组蛋白进行了比较。使用SDS-PAGE、紫外-可见光谱、差示扫描量热法和（1）H-NMR光谱未检测到结构差异。在大肠杆菌中成功表达成熟细胞色素c使得通过定点诱变对cycA基因进行工程改造成为可能，从而为生产用于研究结构/功能关系的突变蛋白提供了一种理想方法。
