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Expression and Purification of Recombinant ADAMTS8
Tina Burkhard1, Alexander Frederick Minns1, Salvatore Santamaria2
1Department of Biochemical Sciences, School of Biosciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, UK.
Methods in Molecular Biology (Clifton, N.J.)
|December 1, 2023
Summary
A Disintegrin-like and Metalloproteinase with Thrombospondin motifs 8 (ADAMTS8) shows anti-cancer properties by inhibiting tumor invasion. This study details methods to produce and purify ADAMTS8 for further research into its cancer-fighting mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- ADAMTS8 (A Disintegrin-like and Metalloproteinase with Thrombospondin motifs 8) is a metalloproteinase downregulated in solid tumors.
- High ADAMTS8 levels in xenografts correlate with reduced tumor invasion and migration.
- The precise anti-cancer mechanisms of ADAMTS8 remain largely unknown.
Purpose of the Study:
- To develop methods for expressing and purifying recombinant ADAMTS8.
- To establish a functional assay for ADAMTS8 proteolytic activity.
- To facilitate further investigation into ADAMTS8's role in cancer.
Main Methods:
- Expression and purification of recombinant ADAMTS8 protein in HEK293T cells.
- Development of an assay to measure ADAMTS8's enzymatic activity.
- Utilized osteopontin (OPN) as a substrate to validate the assay.
Main Results:
- Successfully expressed and purified recombinant ADAMTS8.
- Established a functional assay to detect ADAMTS8 proteolytic activity.
- Demonstrated the cleavage of OPN by purified ADAMTS8.
Conclusions:
- The availability of purified ADAMTS8 and a functional assay enables detailed studies of its biological functions.
- This work provides a foundation for elucidating ADAMTS8's anti-cancerogenic mechanisms.
- Further research can now explore additional substrates and pathways affected by ADAMTS8.
