Novel Cloning Vectors for Stably Expressing NanoBiT® Fusion Proteins

Part # PS331

Abstract

Protein:protein interactions (PPIs) are essential to cellular signal transduction pathways. NanoLuc® Binary Technology (NanoBiT) is a two-subunit system based on NanoLuc® luciferase that can be applied to detecting PPIs. Large BiT (LgBiT; 18 kDa) and Small BiT (SmBiT; 11 amino acid peptide) subunits are expressed as fusions to proteins of interest, where PPI facilitates subunit complementation to give a bright, luminescent enzyme.

Although transient expression of fusion partners can be sufficient for many experiments, stable cell lines are often needed for maximal reproducibility. In this poster, we describe the use of BiBiT-Ready vectors and the BiBiT approach for stable expression of LgBiT and SmBiT fusion proteins. This approach means both fusion proteins are transcribed at similar levels from the same locus, increasing the odds of finding a clone with appropriate levels of expression of both fusion proteins. We demonstrate this approach to make stable cell lines for several key membrane protein interactions: HER2:HER3, VEGFR1:VEGFR1 and CX3CR1/ARRB2. We also describe how to create a stable cell pool for CRAF:BRAF.