Bacterial expression of a mitochondrial cytochrome c.: Trimethylation of Lys72 in yeast iso-1-cytochrome c and the alkaline conformational transition
Bacterial expression of a mitochondrial cytochrome c.: Trimethylation of Lys72 in yeast iso-1-cytochrome c and the alkaline conformational transition
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DOI：
10.1021/bi972188d
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发表时间：
1998-04-28
期刊：
影响因子：
2.9
通讯作者：
Mauk, AG
中科院分区：
文献类型：
--
作者：
Pollock, WBR;Rosell, FI;Mauk, AG
酿酒酵母 iso-1-细胞色素 c 通过编码细胞色素 (CYC1) 和酵母细胞色素 c 血红素裂解酶 (CYC3) 的基因共表达而在大肠杆菌中表达。该表达系统的构建涉及将两个基因平行克隆到载体pUC18中以得到质粒pBPCYC1(wt)/3。转录由位于 CYC1 上游的两个启动子 Lac 和 Trc 指导。两种蛋白均在携带该质粒的大肠杆菌细胞的细胞质中表达。在发酵罐中生长的半厌氧培养物每升培养物产生 15 mg 重组 iso-1-细胞色素 c。通过添加 IPTG 来增加产量的尝试抑制了群体内 CYC1 基因的拷贝数。将大肠杆菌中用 pBPCYC1(wt)/3 表达的野生型 iso-1-细胞色素 c 与酵母中表达的相同蛋白质进行比较。在中性 DH 下，这两种蛋白质表现出无法区分的光谱和物理（T-m、E-m'）特征。然而，电喷雾质谱分析表明，72 位的赖氨酰残基不像酿酒酵母那样被大肠杆菌三甲基化。有趣的是，在大肠杆菌中表达的蛋白质的碱性转变的 pK(a) 类似于在酵母中表达的细胞色素 (8.5-8.7) 中观察到的低 0.6 pK(a) 单位。在高 pH 条件下收集的细菌表达的细胞色素的 H-1 NMR 光谱显示存在第三种碱性构象异构体，而在酵母表达的细胞色素的相应光谱中未观察到该构象异构体。这些观察结果表明，如果 Lys72 未转录后修饰为三甲基赖氨酸，则它可以作为碱性 iso-1-亚铁细胞色素 c 的血红素铁的轴向配体。
Saccharomyces cerevisiae iso-1-cytochrome c has been expressed in Escherichia coli by coexpression of the genes encoding the cytochrome (CYC1) and yeast cytochrome c heme lyase (CYC3). Construction of this expression system involved cloning the two genes in parallel into the vector pUC18 to give the plasmid pBPCYC1(wt)/3. Transcription was directed by two promoters, Lac and Trc, that were located upstream from CYC1. Both proteins were expressed in the cytoplasm of E. coli cells harboring the plasmid. Semianaerobic cultures grown in a fermenter produced 15 mg of recombinant iso-1-cytochrome c per liter of culture, Attempts to increase production by addition of IPTG suppressed the number of copies of the CYC1 gene within the population. Wild-type iso-1-cytochrome c expressed with pBPCYC1(wt)/3 in E. coli was compared to the same protein expressed in yeast. At neutral DH, the two proteins exhibit indistinguishable spectroscopic and physical (T-m, E-m') characteristics. However, electrospray mass spectrometry revealed that the lysyl residue at position 72 is not trimethylated by E. coli as it is by S. cerevisiae. Interestingly, the pK(a) of the alkaline transition of the protein expressed in E. coli is similar to 0.6 pK(a) unit lower than that observed for the cytochrome expressed in yeast (8.5-8.7). H-1 NMR spectroscopy of the bacterially expressed cytochrome collected at high pH revealed the presence of a third alkaline conformer that is not observed in the corresponding spectrum of the cytochrome expressed in yeast. These observations suggest that Lys72 can serve as an axial ligand to the heme iron of alkaline iso-1-ferricytochrome c if it is not modified posttranscriptionally to trimethyllysine.