Binding Rate Screen - a high-throughput assay in soluble lysate for prioritizing protein expression constructs

Area of Science:

• Structural biology
• Protein biochemistry
• High-throughput screening

Background:

• Identifying optimal expression constructs is crucial for structural genomics.
• Current methods for screening protein expression constructs are often labor-intensive and require purified proteins, creating a bottleneck.

Purpose of the Study:

• To develop a novel, high-throughput method for screening protein expression constructs.
• To alleviate the bottleneck in identifying suitable constructs for recombinant protein production.

Main Methods:

• A functional screen termed Binding Rate Screen was developed.
• This method utilizes the hexahistidine (His(6)) tag as a reporter in crude soluble lysate.
• The binding rate of the His(6) tag to an affinity matrix is measured as a proxy for protein quality (aggregation, concentration, purifiability).

Main Results:

• The Binding Rate Screen successfully identifies constructs with high expression of soluble, monomeric protein.
• High binding rates correlate with good protein characteristics, confirmed by analytical size-exclusion chromatography.
• The method prioritizes protein expression constructs in minutes, offering a 10-100x speed improvement over existing technologies.

Conclusions:

• The Binding Rate Screen is a rapid, high-throughput method to overcome bottlenecks in identifying suitable protein expression constructs.
• This functional screening approach accelerates the recombinant protein production pipeline for structural genomics.
• The method enables faster prioritization of protein variants for downstream applications.