Smith M A, Napier J A, Stymne S, Tatham A S, Shewry P R, Stobart A K
Department of Botany, University of Bristol, U.K.
Biochem J. 1994 Oct 1;303 ( Pt 1)(Pt 1):73-9. doi: 10.1042/bj3030073.
Cytochrome b5 from tobacco (Nicotiana tabacum) was expressed in Escherichia coli using a T7 polymerase/promoter system as described by Studier, Rosenberg, Dunn and Dubendorff (1990) (Methods Enzymol. 185, 60-89). Transformed cells were red in colour and accumulated cytochrome b5 to a level of around 30% of the total cell protein. The purified cytochrome had oxidized, reduced and low-temperature absorbance spectra characteristic of plant microsomal cytochrome b5, and exhibited a c.d. spectrum resembling that of a mammalian cytochrome b5. The recombinant protein appeared to be correctly assembled and biologically active, being reduced by NADH in the presence of microsomal membranes prepared from the developing seeds of sunflower (Helianthus annuus). Inhibition of haem synthesis in the transformed E. coli cells expressing cytochrome b5, by the use of gabaculin or succinylacetone, prevented the assembly of the cytochrome b5 holoprotein but had little effect on the accumulation of cytochrome apoprotein. The recombinant protein expressed in E. coli therefore has the biochemical features of the higher-plant cytochrome b5 and can be used in studies of plant microsomal oxidation/reduction reactions.
利用Studier、Rosenberg、Dunn和Dubendorff（1990年）（《酶学方法》185卷，60 - 89页）所述的T7聚合酶/启动子系统，在大肠杆菌中表达了来自烟草（Nicotiana tabacum）的细胞色素b5。转化后的细胞呈红色，细胞色素b5的积累量达到总细胞蛋白的30%左右。纯化后的细胞色素具有植物微粒体细胞色素b5的氧化、还原和低温吸收光谱特征，其圆二色光谱与哺乳动物细胞色素b5的相似。重组蛋白似乎组装正确且具有生物活性，在存在从向日葵（Helianthus annuus）发育种子制备的微粒体膜的情况下，能被NADH还原。通过使用加巴库林或琥珀酰丙酮抑制表达细胞色素b5的转化大肠杆菌细胞中的血红素合成，可阻止细胞色素b5全蛋白的组装，但对细胞色素脱辅基蛋白的积累影响很小。因此，在大肠杆菌中表达的重组蛋白具有高等植物细胞色素b5的生化特性，可用于植物微粒体氧化/还原反应的研究。
