Patel C N, Lind M C, Pielak G J
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Protein Expr Purif. 2001 Jul;22(2):220-4. doi: 10.1006/prep.2001.1438.
We have expressed horse cytochrome c in Escherichia coli. The gene was designed with E. coli codon bias and assembled by using a recursive polymerase chain reaction method. The far-ultraviolet and near-ultraviolet/Soret circular dichroism (CD) spectra show that the structure of recombinant horse cytochrome c is the same as that of the authentic protein. CD-detected thermal denaturation studies were used to measure the thermodynamic parameters associated with two-state denaturation. The free energy of denaturation for the recombinant protein is 10.0 +/- 2.3 kcal mol(-1) at pH 4.6 and 25 degrees C, which agrees with the value for the authentic protein. The expression system will help advance our understanding of the roles of cytochrome c in electron transfer, oxidative stress, and apoptosis by allowing the production of protein variants.
我们已在大肠杆菌中表达了马细胞色素c。该基因按照大肠杆菌密码子偏好性进行设计，并通过递归聚合酶链反应方法进行组装。远紫外和近紫外/索雷特圆二色性（CD）光谱表明，重组马细胞色素c的结构与天然蛋白质相同。利用CD检测的热变性研究来测量与两态变性相关的热力学参数。在pH 4.6和25℃条件下，重组蛋白的变性自由能为10.0±2.3千卡·摩尔⁻¹，这与天然蛋白质的值相符。该表达系统将通过允许生产蛋白质变体，有助于推进我们对细胞色素c在电子传递、氧化应激和细胞凋亡中作用的理解。
