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.
In this study, we applied a highly sensitive small luciferase, NanoLuc, to establish a knock‐in cell line using the CRISPR/Cas9 system and characterized the endogenous promoter activity of the glucose‐regulated protein 78 (GRP78) gene. The N‐terminal region of the human GRP78 gene was fused to the NanoLuc gene and aligned with the puromycin‐resistant gene through the 2A peptide sequence and used as a knock‐in vector. The selected cells responded to both pharmacological and genetic ER stress and show NanoLuc‐based CRISPR/Cas9 system is a very useful tool to isolate gene‐edited cells and to characterize the endogenous promoter activity for genes of interest.
Structure and stability of the gold cluster ions of which skeleton are synthesized as a complex were analyzed using the Hückel method based on graph theory. Hückel Energy (HE) and Topological Resonance Energy (TRE) were determined for neutral, positive ion, and negative ion clusters, where all the isomers of the gold cluster up to octamer were considered. Since some graphically designed isomers include bonds that cannot be realized in three- dimensional space, the screening was carried out by a molecular force field calculation with LAMMPS (lammps.sandia.gov/.). Among the isomers thus obtained, both HE and TRE were most stable when the tetramer was Au4
2 + with a tetrahedral structure, and with the hexamer, Au6
2+ with two tetrahedrons sharing one side. The complexes with these structures have actually been synthesized. On the other hand, the synthesis example of the most stable cluster for octamer Au8 has not been reported.
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