Modifications in the N-terminus of an insect cytochrome P450 enhance production of catalytically active protein in baculovirus-Sf9 cell expression systems.
Modifications in the N-terminus of an insect cytochrome P450 enhance production of catalytically active protein in baculovirus-Sf9 cell expression systems.
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DOI：
10.1016/j.ibmb.2007.09.005
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发表时间：
2008
影响因子：
3.8
通讯作者：
W. Mao;M. Berenbaum;M. Schuler
中科院分区：
文献类型：
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作者：
W. Mao;M. Berenbaum;M. Schuler
尽管杆状病毒载体是在昆虫细胞培养物中异源表达蛋白质的有力工具，但一些昆虫和植物微粒体P450蛋白质在该系统中并未有效表达。假设它们的表达失败可能是由于它们的 N 端序列和相邻胞质序列之间的碰撞造成的，我们将多尾凤蝶 CYP6B33 的 N 端（在 Sf9 细胞中不正确折叠）与其对应的凤蝶 CYP6B1 中存在的序列进行了比较和突变，该序列有效表达并正确折叠。对分隔信号锚结构域 (SAD) 和胞质结构域的接头中的三个差异进行分子建模，确定 CYP6B33 中的 Val32 是对胞质结构域的折叠和/或定位潜在重要的残基。 CYP6B33 连接子（CYP6B33 V32A 突变体）中 Val32 突变为 Ala32 或用 CYP6B1（CYP6B11–20/CYP6B3321–500 突变体）替换 CYP6B33 SAD 允许显着的 P450 表达，表明涉及信号锚和膜连接子的复杂相互作用影响 P450 的折叠和活性。这种异源表达系统。
Although baculovirus vectors are powerful tools for the heterologous expression of proteins in insect cell cultures, some insect and plant microsomal P450 proteins are not effectively expressed in this system. Hypothesizing that their expression failures might result from collisions between their N-terminal sequences and adjacent cytosolic sequences, we compared and mutated the N-terminus of Papilio multicaudatus CYP6B33, which is inappropriately folded in Sf9 cells, to sequences present in its Papilio polyxenes CYP6B1 counterpart, which is efficiently expressed and appropriately folded. Molecular modeling of the three differences in the linker separating the signal anchor domain (SAD) and the cytosolic domain identified Val32 in CYP6B33 as a residue potentially important for folding and/or positioning of the cytosolic domain. Mutation of Val32 to Ala32 in the CYP6B33 linker (CYP6B33 V32A mutant) or replacement of the CYP6B33 SAD with that of CYP6B1 (CYP6B11–20/CYP6B3321–500mutant) allowed for significant P450 expression, indicating that complex interactions involving both the signal anchor and membrane linker affect folding and activity of P450s in this heterologous expression system.