A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

作者：Guo Hou Fu; Cho Eun Jeong; Devkota Ashwini K; Chen Yulong; Russell William; Phillips George N Jr; Yamauchi Mitsuo; Dalby Kevin N^*; Kurie Jonathan M^*

来源：Archives of Biochemistry and Biophysics, 2017, 618: 45-51.

DOI：10.1016/j.abb.2017.02.003

• Lysyl hydroxylase 2
• Collagen
• High-throughput assay
• Succinate detection
• Chinese hamster ovary cell
• Oxygenase

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 alpha-ketoglutarate (alpha KG)-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 C-14-labeled alpha KG 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 aKG to succinate. These methodologies may be applicable to other Fe(II) and alpha KG-dependent oxygenase systems.

• 出版日期2017-3-15