Demonstration of a Simple Epitope Tag Multimerization Strategy for Enhancing the Sensitivity of Protein Detection...

Area of Science:

• Neuroscience
• Molecular Biology
• Genetics

Background:

• Understanding protein expression and distribution is crucial for deciphering neuronal function, including neurotransmitter (NT) usage and release sites.
• Visualizing specific neuronal proteins is challenging due to complex neural networks and low gene expression levels.

Purpose of the Study:

• To develop a method for enhancing the detection sensitivity of neuronal proteins, specifically the *Drosophila* vesicular acetylcholine transporter (vAChT).
• To create conditional alleles of vAChT with multiple epitope tags for improved protein visualization.

Main Methods:

• A novel DNA repeat multimerization strategy using Gibson cloning was serendipitously discovered.
• Optimization yielded alleles with 6-7 copies, and occasionally up to 10 copies, of MYC and OLLAS epitope tag sequences.
• Conditionally expressible genome-edited 7XMYC-vAChT and 6XOLLAS-vAChT variants were generated and characterized.

Main Results:

• The multimerization method successfully generated vAChT alleles with significantly increased epitope tag copies (up to 10x).
• This enhanced sensitivity aids in detecting low-abundance proteins.
• The developed vAChT variants demonstrated conditionality, synaptic vesicle specificity, and neurotransmitter-specific expression.

Conclusions:

• The repeat multimerization technique offers a powerful tool for enhancing protein detection sensitivity in neuroscience research.
• This method is effective for DNA repeats of at least 56 bp and broadly applicable across species.
• The conditional vAChT variants are valuable for cholinergic neurotransmitter phenotyping and defining neuronal polarity, contributing to neural circuit and behavior analysis.