Typically, this is accomplished in mammalian cells by overexpressing recombinant proteins from genetic vectors containing strong transcriptional promoters. (4) Dynamic processes in cells are routinely revealed using genetically encoded protein fusions with reporter tags, most commonly GFP or similar autofluorescent proteins. (3) While antibody detection systems share many of these limitations, dependable quantitation is also limited by the availability of high-quality antibodies. It also has limited dynamic range and struggles with detection of low abundance proteins. Mass spectrometry is relatively low-throughput and usually requires specialized procedures to achieve reliable quantitation. Common approaches for quantifying protein levels and their modifications in complex biological samples include mass spectrometry and antibody-based methodologies, such as ELISA and Western blotting. (1, 2) Elucidating the intrinsic dynamics of these processes can thus offer insight into disease mechanisms and provide predictive models for drug discovery. These results demonstrate the ability to efficiently tag endogenous proteins with a small luminescent peptide, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications.Īs protein expression and post-translational modifications are fundamental to cellular physiology, their disruption can lead to pathophysiological conditions such as cancer, neurodegeneration, and inflammatory diseases. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation. Without requiring clonal isolation of the edited cells, we were able to quantify changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli.
Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation ( K D = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins.
Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications.
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