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A Novel Peptide Tag Enables Simple, Sensitive Bioluminescent Quantification of Tagged Proteins

Part # PS310

Abstract

Christopher Eggers, Braeden Butler, Robin Hurst, Mary Hall, Brock Binkowski, Lance Encell, Marie Schwinn, Thomas Machleidt, Keith Wood & Frank Fan
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI, 53711

Dysregulation of protein expression is a key mechanism of tumorigenesis. The most commonly used approach to monitor changes in expression is to perform SDS-PAGE, followed by immunoblotting, a labor-intensive process that requires high-quality antibodies to detect proteins at endogenous levels of expression. We have developed a novel peptide tag utilizing NanoLuc® Binary Technology (NanoBiT), a binary complementation system based on NanoLuc® luciferase. The tag, designated High BiT (HiBiT), is only 11 amino acids in length, which minimizes potential interference with protein function. The amount of HiBiT-tagged protein is measured using a lytic detection reagent containing Large BiT (LgBiT), which binds with high affinity to HiBiT (KD~1 nM) to reconstitute a bright, luminescent enzyme. HiBiT-tagged proteins can be quantified in cell lysates over 7 orders of magnitude of linear dynamic range with a limit of detection of less than 10-19 moles (3 fg of 30 kDa protein). The simple add-mix-read assay protocol can be completed in minutes, providing an assay that is compatible with high-throughput applications. The sensitivity of the assay allows quantification at endogenous levels of expression, and the small tag size is ideal for CRISPR-mediated genome editing. HiBiT-tagged proteins separated by SDS-PAGE can be detected on blots at sub-picogram levels with a detection reagent containing LgBiT. By eliminating the multiple steps of blocking, binding, and washing of traditional blotting techniques, the protocol takes minutes instead of hours. Additionally, the cell surface expression, internalization, or secretion of HiBiTtagged proteins can be measured in minutes using a non-lytic detection reagent containing cellimpermeable LgBiT and furimazine. HiBiT represents a next-generation protein tag that allows simple quantification of proteins of interest in their cellular context and following SDS-PAGE.

Printed in USA.