Twin Strep-Tag Modified CPT1A Mitochondrial Membrane Chromatography in Screening Lipid Metabolism Regulators

Area of Science:

• Biochemistry
• Chromatography
• Drug Discovery

Background:

• Mitochondrial Membrane Chromatography (MMC) is vital for studying mitochondrial membrane protein-ligand interactions.
• Traditional MMC methods suffer from nonspecific binding due to chemical immobilization.
• Carnitine palmitoyltransferase 1A (CPT1A) is a key mitochondrial enzyme.

Purpose of the Study:

• To develop a highly specific MMC stationary phase (MMSP) for improved ligand screening.
• To establish a Twin Strep-Tag/Strep Tactin affinity system for CPT1A MMSP.
• To identify novel CPT1A ligands from traditional Chinese medicines.

Main Methods:

• Prepared a CPT1A MMSP using Strep-tactin-modified silica gel and Twin Strep-Tag.
• Employed the modified CPT1A/MMC system for ligand screening.
• Conducted biological assessments of identified compounds on HepG2 cells.

Main Results:

• The Twin Strep-Tag/Strep Tactin modified CPT1A/MMC system showed enhanced retention, lifespan, and specificity.
• Identified ononin, isoliquiritigenin, and aloe-emodin as novel CPT1A ligands.
• Screened compounds promoted CPT1A activity, reduced lipid droplets and triglyceride levels in HepG2 cells.

Conclusions:

• Developed a promising MMC system for studying mitochondrial membrane protein bioaffinity.
• The system enables specific bioactive molecule screening from complex herbal extracts.
• This advancement supports mitochondrial medicine discovery and drug development.