The short answer is, you don’t—at least not initially. NanoBRET™ requires that one of your proteins gets tagged with NanoLuc® luciferase while the other protein is tagged with HaloTag® protein. But which protein should receive which tag? And should the tag be placed on the N-terminus or the C-terminus of each protein? If structural information is known, it may seem logical to place a tag at a specific terminus. But these are questions that must be answered empirically. Adding a tag to a protein can affect its ability to fold properly as well as its expression level. It’s also possible that the tag itself could alter or prevent the very interaction you are trying to detect. Therefore, it is recommended to construct a series of eight NanoBRET™ expression vectors incorporating all possible combinations of protein, tag, and terminus.
Full-length Nanos is composed of three distinct regions: N-terminal (N), Zinc Finger (Z) and C-terminal (C). The ZC regions have been shown to be crucial for repression, while the N region only makes a minor contribution. Due to the difficulty in expressing full-length Nanos and Pum, electrophoretic mobility shift assays demonstrating complex formation between Pum and Nanos has been performed with only Nanos ZC and the RNA-binding domain of Pum. So, we chose to use those protein domains in developing our assay.
In our example, we created eight clones of the Drosophila Pum RNA Binding Domain (Pum-RBD) and the Nanos zinc-finger and C-terminal domains (Nanos-ZC) by appending NanoLuc® luciferase (NL or NLuc) donor and HaloTag® (HT) acceptor tags to Pum-RBD and Nanos-ZC using the NanoLuc® NanoBRET™ PPI Flexi® Starter System. Pum-RBD and Nanos-ZC were first cloned into the pFN21A (N-terminal HaloTag®) vector and then additional clones with the proper tags at each orientation were transferred from the pFN21A vectors using Flexi® cloning. Before proceeding to the assay, each clone was sequenced to ensure that the correct DNA sequence was present in-frame with no mutations introduced. Clones were also created for full-length Nanos. Results with the Nanos-ZC were found to be similar to full-length Nanos, so we show only data with Nanos-ZC here.