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

(1)研究了氨基末端结构对细胞色素p450在大肠杆菌细胞中的表达水平的影响。n端序列包含三个特征片段；具有I型信号锚定序列（SA-I）、碱性残基富序列（BS）和脯氨酸富区（PR）功能的疏水序列。sa - 1和BS能在P450分子种间交换，而PR不能。特定的同工异构体需要其特定的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起关键作用。(2)使用其他P450分子，线粒体和细菌可溶性P450也证明了PR对正确折叠的要求。因此，我们得出结论，PR对所有细胞色素P450同工型的正确折叠至关重要。(3)阐明了sa - 1膜蛋白在内质网膜上的拓扑形成过程。(4)明确了线粒体外膜sa - 1蛋白的靶向基序。(5) nadph -细胞色素P450还原酶也具有sa - 1序列。(6)我们还提出了几条证据，证明sa - 1在包括多跨膜蛋白在内的各种膜蛋白的整合中起关键作用。

(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.