Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated using E coli– or insect-based expression systems is either insoluble or enzymatically unstable, and the LH2 enzymatic activity assays that are currently available measure radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems.
|Number of pages||7|
|Journal||Archives of Biochemistry and Biophysics|
|State||Published - Mar 15 2017|
Bibliographical noteFunding Information:
This work was supported by NIH grants P50-CA70907-15 (JMK) and R01 CA105155 (JMK, MY), the Welch Foundation (F-1390) (KND), and CPRIT grants RP160652 (JMK, GNP), RP160657 (KND), and RP110532-P1 (KND).
© 2017 Elsevier Inc.
- Chinese hamster ovary cell
- High-throughput assay
- Lysyl hydroxylase 2
- Succinate detection
ASJC Scopus subject areas
- Molecular Biology