Enhanced recombinant expression and purification of human IRAP for biochemical and crystallography studies.
Enhanced recombinant expression and purification of human IRAP for biochemical and crystallography studies.
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用于生化和结晶学研究的人IRAP的高效重组表达和纯化。
DOI：
10.1016/j.bbrep.2021.101042
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发表时间：
2021-09
影响因子：
2.7
通讯作者：
Guo HC
中科院分区：
文献类型：
--
作者：
Sui L;Guo HC
胰岛素调节氨基肽酶(IRAP)是一种膜结合酶，具有多种功能。它最初被描述为胰岛素反应葡萄糖转运体GLUT4的伴侣蛋白，在专门的囊泡中。该蛋白随后被证明与催产素酶/氨基肽酶或血管紧张素IV(Ang IV)受体(AT4受体)相同。一些AT4配体多肽，如血管紧张素转换酶IV和LVV-血吗啡-7，已被证明是IRAP的抑制剂，具有增强记忆的特性。因此，IRAP一直是开发认知增强剂的目标。为了便于对IRAP的催化和抑制进行详细的机理研究，并为IRAP与多肽抑制剂的复合体的生物物理和结构研究铺平道路，我们报道了一种利用High Five昆虫细胞的优化表达和纯化系统。我们还报道了纯化的重组IRAP的生化性质，该重组IRAP含有标准的氨基肽酶底物和一个优化的IRAP多肽抑制剂，Ki值为98nM。
Insulin-regulated aminopeptidase (IRAP) in humans is a membrane bound enzyme that has multiple functions. It was first described as a companion protein of the insulin-responsive glucose transporter, Glut4, in specialized vesicles. The protein has subsequently been shown to be identical to the oxytocinase/aminopeptidase or the angiotensin IV (Ang IV) receptor (AT4 receptor). Some AT4 ligand peptides, such as Ang IV and LVV-hemorphin-7, have been shown to act as IRAP inhibitors that exert memory-enhancing properties. As such IRAP has been a target for developing cognitive enhancers. To facilitate detailed mechanistic studies of IRAP catalysis and inhibition, and to pave the way for biophysical and structural studies of IRAP in complex with peptide inhibitors, we report here an optimized expression and purification system using High Five insect cells. We also report biochemical characterizations of the purified recombinant IRAP with a standard aminopeptidase substrate and an optimized IRAP peptide inhibitor with a Ki of 98 nM.
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影响因子：
4.7
作者：
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通讯作者：
Albiston, AL
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通讯作者：
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作者：
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