Application of a novel HiBiT peptide tag for monitoring ATF4 protein expression in Neuro2a cells.
Application of a novel HiBiT peptide tag for monitoring ATF4 protein expression in Neuro2a cells.
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DOI：
10.1016/j.bbrep.2017.08.002
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发表时间：
2017-12
影响因子：
2.7
通讯作者：
Hirata Y
中科院分区：
文献类型：
--
作者：
Oh-Hashi K;Furuta E;Fujimura K;Hirata Y
开发了一种由两个片段（大n端和小c端区域（NanoBiT））组成的分裂NanoLuc检测系统，用于研究活细胞内蛋白质-蛋白质相互作用。有趣的是，在11个c端氨基酸中替换5个氨基酸显著增加了对大n端片段LgBiT的亲和力，并且该复合物具有NanoLuc荧光素酶活性。在本研究中，我们首先利用这个小片段HiBiT，通过短暂过表达HiBiT标记的ATF4来阐明ATF4蛋白的表达。根据ATF4内在蛋白的调节，通过检测细胞裂解液和SDS-PAGE后的荧光素酶活性，并将其转移到PVDF膜上，观察hibitt标记的ATF4与蛋白酶体抑制剂MG132的稳定。接下来，我们使用CRISPR/Cas9系统将hibit表位标签敲入ATF4基因，并通过测量每个细胞悬液中荧光素酶的活性快速选择阳性克隆。通过一个选择的克隆，我们观察到在选择的细胞中，hibit标记的ATF4的表达随着蛋白质合成抑制剂或蛋白酶体抑制剂和tunicamycin的处理而变化。总之，这种新颖的HiBiT标签是评估感兴趣的蛋白质内源性表达水平的有用工具。一种新的小表位标签HiBiT被用于检测ATF4蛋白的表达。利用CRISPR/Cas9系统生成ATF4-HiBiT Neuro2a细胞。这种HiBiT标签是研究其他蛋白质的有用工具。
A split NanoLuc assay system consisting of two fragments, large N-terminal and small C-terminal regions (NanoBiT), was developed to investigate protein-protein interactions within living cells. Interestingly, the replacement of five amino acids among 11 C-terminal amino acids dramatically increased affinity against the large N-terminal fragment, LgBiT, and the complex had NanoLuc luciferase activity. In this study, we first applied this small fragment, HiBiT, to elucidate the expression of ATF4 protein by transient overexpression of HiBiT-tagged ATF4. According to the regulation of intrinsic ATF4 protein, stabilization of HiBiT-tagged ATF4 with a proteasome inhibitor, MG132, was observed by detecting luciferase activity in cell lysate and after SDS-PAGE and transfer onto a PVDF membrane. Next, we knocked-in the HiBiT-epitope tag into the ATF4 gene using the CRISPR/Cas9 system and rapidly selected positive clones by measuring luciferase activity in an aliquot of each cell suspension. Using a selected clone, we observed that the expression of HiBiT-tagged ATF4 in the selected cells varied in response to treatment with protein synthesis inhibitors or proteasome inhibitors and tunicamycin. Altogether, this novel HiBiT tag is a useful tool to evaluate the endogenous expression levels of proteins of interest. A novel small epitope-tag, HiBiT, was applied to detect ATF4 protein expression. ATF4-HiBiT Neuro2a cells were generated with CRISPR/Cas9 system. This HiBiT tag is a useful tool to study other proteins.