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

Hou Fu Guo, Eun Jeong Cho, Ashwini K. Devkota, Yulong Chen, William Russell, George N. Phillips, Mitsuo Yamauchi, Kevin N. Dalby, Jonathan M. Kurie

Research output: Contribution to journalArticle

4 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)45-51
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume618
DOIs
StatePublished - Mar 15 2017

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Keywords

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Guo, H. F., Cho, E. J., Devkota, A. K., Chen, Y., Russell, W., Phillips, G. N., Yamauchi, M., Dalby, K. N., & Kurie, J. M. (2017). A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay. Archives of Biochemistry and Biophysics, 618, 45-51. https://doi.org/10.1016/j.abb.2017.02.003