Karim A, Kaderbhai N, Evans A, Harding V, Kaderbhai M A
Department of Biochemistry, School of Life Sciences, University of Wales, Aberystwyth, U.K.
Biotechnology (N Y). 1993 May;11(5):612-8. doi: 10.1038/nbt0593-612.
The soluble core domain of cytochrome b5 of liver endoplasmic reticulum was appended at its amino terminus to full-length alkaline phosphatase secretory signal sequence including the ribosomal binding site. The chimeric precursor gene was placed under the transcriptional control of the native pho promoter in a prokaryotic expression vector. Induction of Escherichia coli by growth in a phosphate-limited medium resulted in abundant synthesis of cytochrome b5 as detected spectrophotometrically and by visual transformation of the bacteria to a pink color. The signal-appended cytochrome b5, but not the corresponding signal-deficient derivative, was translocated across the bacterial inner membrane and processed to yield authentic, haem-assembled cytochrome b5 within the periplasm. The eventual processing of the chimeric cytochrome b5 precursor was unusual regarding the known reaction specificity of signal peptidase. The exported, mature haemoprotein was biochemically indistinguishable from its native mammalian counterpart. At peak induction, approximately 6 mg of correctly matured cytochrome b5 per liter of culture was exported. This amount of cytochrome b5 constituted 6% (w/w) of the periplasmic protein. The appearance of the exported apo-cytochrome b5 preceded the formation of holo-protein. Thus the eukaryotic cytoplasmic protein was efficiently exported from E. coli and post-translocationally modified to generate a functional haemoprotein in the periplasm.
肝脏内质网细胞色素b5的可溶性核心结构域在其氨基末端与包含核糖体结合位点的全长碱性磷酸酶分泌信号序列相连。嵌合前体基因置于原核表达载体中天然pho启动子的转录控制之下。在磷酸盐限制培养基中生长诱导大肠杆菌，通过分光光度法检测以及观察到细菌变为粉红色，结果显示细胞色素b5大量合成。附加信号的细胞色素b5，而非相应的信号缺失衍生物，穿过细菌内膜并被加工，从而在周质中产生真正的、结合血红素的细胞色素b5。就信号肽酶已知的反应特异性而言，嵌合细胞色素b5前体的最终加工过程不同寻常。输出的成熟血红蛋白在生化性质上与其天然哺乳动物对应物无法区分。在诱导高峰期，每升培养物中约有6毫克正确成熟的细胞色素b5被输出。这个量的细胞色素b5占周质蛋白的6%（w/w）。输出的脱辅基细胞色素b5的出现先于全蛋白的形成。因此，真核细胞质蛋白从大肠杆菌中高效输出，并在转运后被修饰，从而在周质中产生一种功能性血红蛋白。
