Selecting optimum eukaryotic integral membrane proteins for structure determination by rapid expression and...

Area of Science:

• Biochemistry
• Structural Biology
• Proteomics

Background:

• Determining membrane protein structures is crucial for understanding cellular functions.
• Existing methods often face challenges in expressing and purifying sufficient quantities of membrane proteins for structural analysis.

Purpose of the Study:

• To establish a medium-throughput pipeline for identifying membrane proteins amenable to structural determination.
• To expand the knowledge of membrane protein structures through proteome-wide coverage.

Main Methods:

• Rationally selected 351 membrane proteins from Saccharomyces cerevisiae for homologous expression.
• Performed medium-scale expression and solubilization tests using n-dodecyl-beta-D-maltopyranoside.
• Demonstrated pipeline extensibility with expression of human solute carrier superfamily proteins.

Main Results:

• Successfully expressed 272 out of 351 tested Saccharomyces cerevisiae membrane proteins.
• 234 of the expressed proteins were solubilized, indicating suitability for further purification.
• Identified a subset of proteins purified to homogeneity for structure determination.

Conclusions:

• The developed pipeline efficiently identifies membrane proteins suitable for structural biology.
• This approach facilitates proteome-wide exploration of membrane protein structures.
• The method is adaptable for expressing membrane proteins from various eukaryotic organisms, including humans.