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

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

SUBMITTER: Guo HF 

PROVIDER: S-EPMC5619865 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature