Development of the Expression System for Cytochrome P450 Based on the Folding Processes of Membrane Proteins

(1)我们研究了大肠杆菌细胞色素P450的氨基端结构对表达水平的影响。N-末端序列包含三个特征性区段;作为I型信号锚定序列（SA-1）的疏水序列、碱性残基富集序列（BS）和脯氨酸富集区（PR）。SA-Ⅰ和BS可在P450分子间交换，PR则不能。特定的同种型需要其特定的PR序列。PR可以从折叠的分子中去除，表明该片段仅在折叠过程中是关键的。(2)PR对正确折叠的要求也使用其他P450分子、线粒体和细菌可溶性P450来证明。因此，我们得出结论，PR是所有细胞色素P450亚型的正确折叠的关键。(3)阐明了SA-Ⅰ膜蛋白在内质网膜上的定位过程。(4)我们明确了线粒体外膜SA-Ⅰ蛋白正确定位的靶向基序。(5)NADPH-细胞色素P450还原酶也具有SA-Ⅰ序列。(6)我们还提出了一些证据表明，SA-Ⅰ在包括多跨膜蛋白在内的各种膜蛋白的整合中起着关键作用。

(1) We have investigated how the amino- (N) -terminal structure affected the expression level of cytochrome P450s in the E coli cells. The N-terminal sequence contains three characteristic segments ; hydrophobic sequence that functions as a type I signal-anchor sequence (SA-I), basic residues-rich sequence (BS), and proline rich region (PR). SA-I and BS could be exchanged among molecular species of P450, whereas PR could not. Specific isoform requires its specific PR sequence. The PR can be removed from folded molecule, indicating that this segment is critical only during the folding process. (2) The requirement of PR for correct folding was also demonstrated using other P450 molecules, mitochondrial and bacterial soluble P450s. Thus we concluded that PR is critical for correct folding of all cytochrome P450 isoforms. (3) We clarified topogenic process of the SA-I membrane proteins on the ER membrane. (4) We clarified targeting motif for the correct location of the SA-I protein of mitochondrial outer membrane. (5) NADPH-cytochrome P450 reductase possesses also the SA-I sequence. (6) We also presented the several lines of evidence demonstrating that SA-I plays a critical role for integration of various membrane proteins including the multispanning membrane proteins.