Expression, Purification, and Functional Analysis Methods of Antigen-Processing Aminopeptidases ERAP1, ERAP2, and
1Laboratory of Biochemistry, Department of Chemistry, National and Kapodistrian University of Athens, Athens, Greece. ampakali@gmail.com.
Methods in Molecular Biology (Clifton, N.J.)
|January 1, 2026
Summary
This study details methods for producing and testing endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1, ERAP2) and Insulin-Regulated Aminopeptidase (IRAP). These enzymes are crucial for immune regulation and disease research, and these protocols enable their study.
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1, ERAP2) and Insulin-Regulated Aminopeptidase (IRAP) are critical for antigen processing and adaptive immune responses.
- Dysregulation of ERAP1, ERAP2, and IRAP is linked to autoimmune diseases and cancer.
Purpose of the Study:
- To provide a comprehensive methodological guide for the expression, purification, and functional characterization of recombinant ERAP1, ERAP2, and IRAP.
- To enable robust biochemical, structural, mechanistic, and drug discovery studies involving these key enzymes.
Main Methods:
- Baculovirus-mediated expression in insect cells for recombinant protein production.
- Affinity chromatography for high-purity enzyme isolation.
- Fluorogenic, colorimetric, and HPLC-based assays for functional characterization, including enzymatic activity, substrate specificity, and inhibitor screening.
Main Results:
- Detailed protocols yielding high-purity, functionally active ERAP1, ERAP2, and IRAP.
- Versatile assay suite for comprehensive enzymatic analysis and inhibitor evaluation.
- Established methods supporting further research into enzyme mechanisms and therapeutic development.
Conclusions:
- The provided protocols facilitate the production and characterization of essential enzymes involved in immune regulation.
- These methods are vital for advancing research in autoimmune disorders, cancer, and drug discovery targeting ERAP1, ERAP2, and IRAP.
Keywords:
Affinity chromatographyAminopeptidase enzymatic assaysAntigen processingBaculovirus-mediated expressionEndoplasmic reticulum aminopeptidases (ERAP1, ERAP2)Enzyme kineticsInhibitor screeningInsulin-regulated aminopeptidase (IRAP)Recombinant protein expressionMore Related Videos
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