Expression, purification and stabilization of human serotonin transporter from E. coli

Area of Science:

• Neuroscience
• Structural Biology
• Biochemistry

Background:

• The serotonin transporter (SERT) is a key drug target for treating depression.
• Recent structural studies of human SERT (hSERT) are crucial for antidepressant development.
• Large-scale protein production for structural analysis of hSERT remains a significant challenge.

Purpose of the Study:

• To establish a robust method for producing purified hSERT in E. coli.
• To identify factors that enhance hSERT stability for structural and functional studies.
• To characterize the binding kinetics of an inhibitor to purified hSERT.

Main Methods:

• Expression and purification of hSERT in E. coli using Fos-choline 12.
• Circular Dichroism (CD) spectroscopy to assess protein stability with various additives.
• Kinetic analysis of inhibitor binding using surface plasmon resonance (SPR) or similar techniques.

Main Results:

• Obtained purified hSERT with >95% purity and a yield of 1.2 mg/L.
• Identified CHS and POPX as stabilizers, increasing hSERT thermostability by 7°C.
• Determined the dissociation constant (KD) for Desipramine as 2.8 μM, with specific association (ka) and dissociation (kd) rates.

Conclusions:

• Developed an efficient E. coli-based system for producing high-purity hSERT.
• Discovered stabilizing agents that improve hSERT stability, facilitating further structural studies.
• Provided key kinetic data for the interaction between hSERT and Desipramine, relevant for drug design.