Profiling Interactome Networks with the HaloTag-NAPPA In Situ Protein Array

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Protein arrays are high-throughput methods for evaluating molecular interactions at the proteome scale.
• Conventional protein arrays can be labor-intensive, time-consuming, and costly.
• Efficient screening of protein-protein interactions (PPIs) is crucial for understanding cellular functions.

Purpose of the Study:

• To develop a modified in situ protein array, the nucleic acid programmable protein assay (NAPPA), for cost-effective and efficient screening of thousands of open reading frames (ORFs).
• To describe the fabrication and application of the HaloTag-NAPPA protein array for detecting protein-protein interactions (PPIs).
• To demonstrate the utility of this array system for generating a protein interactome map.

Main Methods:

• Fabrication of the HaloTag-NAPPA protein array using publicly available resources.
• In situ protein expression on a glass slide utilizing the Halo high-affinity capture tag.
• Detection of protein-protein interactions (PPIs) through the developed array system.

Main Results:

• The HaloTag-NAPPA protein array enables efficient in situ protein capture and expression.
• The developed array facilitates the detection of protein-protein interactions (PPIs).
• The protocol allows for the generation of a protein interactome map.

Conclusions:

• The HaloTag-NAPPA system offers a lower-cost, less labor-intensive, and faster alternative to conventional protein arrays for high-throughput screening.
• This method is effective for detecting protein-protein interactions and constructing protein interactome maps.
• The described protocols provide a foundation for utilizing NAPPA for large-scale proteomic studies.