Protein Pull-down Assay Using HiBiT-tag-dependent Luciferase Activity Measurement

Background

Most proteins interact with their partner proteins to carry out their biological activity. To understand the role of proteins in cells, co-immunoprecipitation (co-IP) or pull-down assays are frequently used to characterize protein–protein interactions. Western blotting (WB) is commonly used to detect prey proteins in co-IP or pull-down assays. However, its sensitivity is low or dependent on the primary antibody, and it gives inaccurate quantification.

High Bit peptide of NanoLuc Binary Technology (NanoBiT) (HiBiT-tag, amino acid sequence: VSGWRLFKKIS) is a part of split NanoLuc luciferase that can reconstitute intact NanoLuc luciferase when another part of split NanoLuc luciferase, Large Bit peptide of NanoBiT (LgBiT), is present. Therefore, the addition of recombinant LgBiT protein and NanoLuc luciferase substrate and measurement of luminescence facilitates the detection of HiBiT-tag (Dixon et al., 2016). Since HiBiT-tag is a short peptide tag with a length of 11 amino acids, it has a minimum effect on the function of fused proteins. Furthermore, a HiBiT-tag-dependent NanoLuc luciferase detection system is useful for quantifying small quantities of protein, because the signal-to-noise ratio is significantly high in NanoLuc luciferase–dependent luminescence, which allows for small-scale experiments. The procedure is simple and therefore suitable for high-throughput assays. In this report, we introduce the method of using the HiBiT-tag-dependent NanoLuc luciferase system for the detection of prey protein in a pull-down assay. Generally, proteins form an oligomeric complex with various partners. In these cases, the depletion of key subunits significantly affects the formation of the entire complex. In this report, we demonstrate that depletion of a mediator protein affects ternary complex formation.

Japanese encephalitis virus (JEV), a single-stranded positive-sense RNA virus, is a human pathogenic flavivirus. In JEV-infected cells, endoplasmic reticulum membrane–derived large compartments (also called viral replication organelles) are observed. Viruses have been considered to efficiently replicate in these compartments, which may be a target for the development of antiviral reagents (Arakawa and Morita, 2019). Previously, our group reported that the host factor valosin-containing protein (VCP) is recruited to the viral replication organelle and helps in viral genome replication (Tabata et al., 2021). However, no direct interaction between VCP and viral proteins were detected. Our previous study revealed a mediator that bridges the interaction between them. We found that the interaction of nuclear protein localization protein 4 (NPL4), a VCP-associating co-factor, and NS4B, a nonstructural viral protein that localizes on the viral replication site, is important for the recruitment of VCP to the viral replication organelle. We have shown that depletion of NPL4 via siRNA knockdown significantly reduces the affinity between VCP and NS4B, through pull-down assay utilizing HiBiT-tag-dependent luciferase activity (HiBiT activity) measurement (Arakawa et al., 2022) (Figure 1A). HiBiT-tagged NS4B and One-Strep-FLAG (OSF)-tagged VCP were co-expressed in 293T cells and the VCP were affinity-purified using Strep-Tactin beads (Figure 1B). The amount of NS4B in the VCP-bound fraction was then determined in the presence of NPL4 and compared with that in the absence of NPL4 by measuring HiBiT activity (Figure 2C). This protocol is not only useful for studying virus–host interactions, but also has broader applications in the investigation of general protein–protein interactions.