3 research outputs found
CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide
Intracellular
signaling pathways are mediated by changes in protein
abundance and post-translational modifications. A common approach
for investigating signaling mechanisms and the effects induced by
synthetic compounds is through overexpression of recombinant reporter
genes. Genome editing with CRISPR/Cas9 offers a means to better preserve
native biology by appending reporters directly onto the endogenous
genes. 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. HiBiT is a 1.3 kDa peptide (11 amino
acids) capable of producing bright and quantitative luminescence through
high affinity complementation (<i>K</i><sub>D</sub> = 700
pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). 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.
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. In combination with fluorescent antibodies,
we further used HiBiT to directly correlate HIF1α levels with
the hydroxyproline modification that mediates its degradation. 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
CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide
Intracellular
signaling pathways are mediated by changes in protein
abundance and post-translational modifications. A common approach
for investigating signaling mechanisms and the effects induced by
synthetic compounds is through overexpression of recombinant reporter
genes. Genome editing with CRISPR/Cas9 offers a means to better preserve
native biology by appending reporters directly onto the endogenous
genes. 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. HiBiT is a 1.3 kDa peptide (11 amino
acids) capable of producing bright and quantitative luminescence through
high affinity complementation (<i>K</i><sub>D</sub> = 700
pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). 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.
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. In combination with fluorescent antibodies,
we further used HiBiT to directly correlate HIF1α levels with
the hydroxyproline modification that mediates its degradation. 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
NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells
Protein-fragment
complementation assays (PCAs) are widely used
for investigating protein interactions. However, the fragments used
are structurally compromised and have not been optimized nor thoroughly
characterized for accurately assessing these interactions. We took
advantage of the small size and bright luminescence of NanoLuc to
engineer a new complementation reporter (NanoBiT). By design, the
NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly
associate so that their assembly into a luminescent complex is dictated
by the interaction characteristics of the target proteins onto which
they are appended. To ascertain their general suitability for measuring
interaction affinities and kinetics, we determined that their intrinsic
affinity (<i>K</i><sub>D</sub> = 190 μM) and association
constants (<i>k</i><sub>on</sub> = 500 M<sup>–1</sup> s<sup>–1</sup>, <i>k</i><sub>off</sub> = 0.2 s<sup>–1</sup>) are outside of the ranges typical for protein interactions.
The accuracy of NanoBiT was verified under defined biochemical conditions
using the previously characterized interaction between SME-1 β-lactamase
and a set of inhibitor binding proteins. In cells, NanoBiT fusions
to FRB/FKBP produced luminescence consistent with the linear characteristics
of NanoLuc. Response dynamics, evaluated using both protein kinase
A and β-arrestin-2, were rapid, reversible, and robust to temperature
(21–37 °C). Finally, NanoBiT provided a means to measure
pharmacology of kinase inhibitors known to induce the interaction
between BRAF and CRAF. Our results demonstrate that the intrinsic
properties of NanoBiT allow accurate representation of protein interactions
and that the reporter responds reliably and dynamically in cells