In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish.
In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish.
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DOI：
10.1002/0471140864.ps2916s84
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发表时间：
2016-04-01
影响因子：
--
通讯作者：
Egli M
中科院分区：
文献类型：
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作者：
Lei L;Egli M
鱼和人细胞色素P450（P450）17 A1催化类固醇17α-羟基化和17α，20-裂解酶反应。Fish P450 17 A2仅催化17α-羟基化。这两种酶都是微粒体型P450，是通过其N-末端疏水片段（信号锚序列）与膜结合的完整膜蛋白。该N-末端区域的存在使得全长蛋白质的表达不可能或具有挑战性。对于某些蛋白质，信号锚序列的可变截短阻止表达或导致表达水平差。为了使P450 17 A1和17 A2结晶以深入了解它们的不同活性，我们使用了替代的N-末端序列来促进表达以及原位蛋白水解。我们的方法，以确定可结晶的P450片段的关键特征是使用的N-末端前导序列，筛选组成的12种蛋白酶，以建立最佳的切割，蛋白酶浓度的变化与SDS-PAGE分析相结合，并使用Edman测序分析所得的片段。本单元描述了两种膜结合P450蛋白的载体制备、表达、纯化和原位蛋白水解结晶的方案。
Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins impossible or challenging. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins.