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

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Guo HF, Cho EJ, Devkota AK, Chen Y, Russell W, Phillips GN Jr, Yamauchi M, Dalby KN, Kurie JM

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

Arch Biochem Biophys. 2017 Mar 15;618:45-51. doi: 10.1016/j.abb.2017.02.003. Epub 2017 Feb 20.

PubMed ID

28216326 [ View in PubMed

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

DrugBank Data that Cites this Article

Drug Enzymes

Drug	Enzyme	Kind	Organism	Pharmacological Action	Actions
Succinic acid	Procollagen-lysine,2-oxoglutarate 5-dioxygenase 3	Protein	Humans	Unknown	Product of	Details