Accurately and quantitatively monitoring endogenous protein expression and dynamics is a core requirement in modern drug discovery and mechanism research. The HiBiT-KI system introduces an 11-amino acid HiBiT tag into the target protein, which, when combined with the LgBiT complement and substrate, forms a stable NanoBiT® luciferase for highly sensitive quantification from live cells to lysates. This document uses multiple HiBiT-KI cell lines to detail technical advantages and application scenarios.
HiBiT Tag Principle: HiBiT is an 11-amino-acid microtag that binds LgBiT with high affinity. The resulting complex forms an active NanoBiT enzyme, producing a luminescent signal in the presence of substrate.
HiBiT Knock-In Method: Using CRISPR/Cas9-mediated homologous recombination, the HiBiT sequence is precisely integrated at the C-terminus of the target gene, preserving endogenous expression and protein function.
Once HiBiT is knocked in, the luminescent signal directly reflects changes in endogenous protein expression. Since it’s an endogenous knock-in, it authentically reflects intracellular protein degradation and stability dynamics, offering a reliable platform to study degradation mechanisms.
HiBiT-KI Technology Applications:
- Oncogenic Mutations: KRAS-G12C/G12D/G13D (MIAPaCa2, HCT116-HiBiT) — Real-time monitoring of KRAS half-life to evaluate the effect of covalent inhibitors on the stability of mutant KRAS.
- Metabolic Regulation: LDHA (293T-HiBiT) — Dynamic tracking of degradation curves under FX11-derived PROTAC treatment; high-throughput screening of metabolic reprogramming inhibitors.
- Epigenetic Modulation: EZH2, PRMT5 (multi-line HiBiT) — Real-time capture of degradation rates of histone methyltransferases under inhibitor treatment, analyzing links between epigenetic regulation and drug resistance.
- Degrader Evaluation: GSPT1 (293T-HiBiT, molecular glue CC-885), BTK (TMD8-HiBiT, PROTACs NX-2127/NX-5948) — Comparison of different degraders’ DC₅₀ and kinetics; luminescent signal directly shows efficiency differences.
HiBiT-KI cell lines, with their speed, quantifiability, and wide dynamic range, are becoming key platforms for target function studies and small molecule drug discovery. They authentically reveal the dynamics of endogenous protein expression and degradation, providing full-process acceleration for mechanism research and candidate evaluation.
HiBiT Products and Services List:
- Ready-to-Use HiBiT-KI Reporter Cell Lines: KRAS-G12C, KRAS-G12D, KRAS-G13D, LDHA, GSPT1, IRAK3, ELAVL1, GRK2, VAV1, PRMT5, BTK, EZH2, FYN, STAT3α/β, ESR1, AR, KRT80, BRD4, IKZF3, etc.
- Custom Knock-In Services: Endogenous knock-in of HiBiT, NanoLuc, HaloTag, etc., into any cell line.
- Phenotype/Function Assays: Luminescence quantification, protein degradation kinetics, rapid PPI screening.
- Data Analysis Support: Dose–response curve plotting, IC₅₀ calculation, and mechanism validation.
Case Studies:
