High-Throughput Protein Expression Screening of Cell-Surface Protein Ectodomains

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Cell-surface receptors are crucial for cellular communication but challenging to express and purify for studies.
• Recombinant ectodomain expression is preferred for large-scale production, necessitating the design and testing of truncation constructs.
• Individual testing of numerous protein constructs is labor-intensive and time-consuming.

Purpose of the Study:

• To develop a high-throughput method for rapidly assessing the expression of multiple recombinant cell-surface receptor ectodomain constructs simultaneously.
• To streamline the identification of well-expressing constructs for efficient large-scale protein production.
• To reduce the time and resources required for optimizing protein expression for structural and biochemical studies.

Main Methods:

• A high-throughput ELISA fluorescence assay was developed for rapid screening.
• Cell-surface ectodomains were expressed at small scale and enzymatically biotinylated.
• Detection was achieved using a C-terminal His-tag, enabling simultaneous assessment of numerous constructs.

Main Results:

• The small-scale ELISA successfully prioritized well-expressing constructs for the neurexin, neuroligin, and latrophilin families.
• The method demonstrated efficiency in detecting clones with low expression levels.
• The approach significantly streamlined the process of identifying optimal protein candidates.

Conclusions:

• The developed high-throughput ELISA fluorescence assay is an effective tool for rapidly assessing recombinant ectodomain expression.
• This method significantly accelerates the identification of suitable protein constructs for large-scale production.
• The approach offers a valuable solution for overcoming challenges in cell-surface receptor research, saving time and resources.