Acetylcholinesterase assay for rapid expression screening in liquid and solid media

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Acetylcholinesterase (AChE) is a key enzyme in synaptic function, implicated in Alzheimer's disease, and a target for insecticides and warfare agents.
• Recombinant AChE production is crucial for research, with the yeast Pichia pastoris being a promising but improvable expression system.
• Efficient screening methods are needed to identify AChE-expressing yeast colonies for optimizing recombinant protein production.

Purpose of the Study:

• To develop a rapid and efficient chromogenic screening test for detecting acetylcholinesterase (AChE) activity in Pichia pastoris.
• To enable high-throughput screening of yeast colonies for improved recombinant AChE expression.
• To facilitate the optimization of AChE production for applications in biosensors and therapeutic research.

Main Methods:

• A chromogenic substrate, indoxylacetate, was utilized to detect AChE activity.
• The assay was designed to be insensitive to background color from yeast culture media.
• The test was adapted for both microplate screening and direct on-plate detection of positive colonies.

Main Results:

• A novel chromogenic assay was successfully developed for detecting AChE activity in yeast.
• The assay demonstrated suitability for microplate screening, overcoming background color interference.
• The test was effective for direct on-plate identification of AChE-expressing colonies, simplifying screening.

Conclusions:

• The developed chromogenic test provides a rapid, efficient, and reliable method for screening AChE-expressing Pichia pastoris colonies.
• This screening tool is valuable for optimizing recombinant AChE production, supporting its use in biosensors and disease research.
• The assay's adaptability for microplate and on-plate detection enhances its utility in biotechnology workflows.