N-terminal truncation circumvents proteolytic degradation of the human HtrA2/Omi serine protease in Escherichia coli: Rapid purification of a proteolytically active HtrA2/Omi

Young Mo Seong, Hyo Jin Park, Geun Hye Seong, Ju Youn Choi, Sung Joo Kim Yoon, Byung Re Min, Seongman Kang, Hyangshuk Rhim

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)

Abstract

HtrA2/Omi, a mitochondrial trypsin-like serine protease, is pivotal in regulating apoptotic cell death; however, the underlying mechanism of HtrA2/Omi-mediated apoptosis remains to be elucidated. Using the pGEX bacterial expression system, we investigated the expression patterns of various forms of HtrA2/Omi. Full-length mouse HtrA2/Omi (mHtrA2/Omi) was successfully expressed in E. coli and purified as a proteolytically active protein. In contrast, the expression of full-length human HtrA2/Omi (hHtrA2/Omi) in E. coli was barely detected. On the basis of this result, we characterized further the expression patterns of N- or C-terminally truncated hHtrA2/Omi proteins. We found that three copies of the PRAXXTXXTP motif, which exist only in hHtrA2/ Omi, might serve as a primary site that is highly susceptible to proteolytic degradation by host proteases. Removal of the N-terminal region containing the PRAXXTXXTP motifs produced a form resistant to proteolytic degradation during expression in E. coli and purification, consequently improving the production of a catalytically active, mature hHtrA2/Omi. Our study provides a method for generating useful reagents to investigate molecular mechanism by which HtrA2/ Omi contributes to regulating apoptotic cell death and to identify natural substrates of HtrA2/Omi.

Original languageEnglish
Pages (from-to)200-208
Number of pages9
JournalProtein Expression and Purification
Volume33
Issue number2
DOIs
Publication statusPublished - 2004 Feb

Keywords

  • Mitochondrial serine protease
  • PRAXXTXXTP motif
  • Proteolytic degradation
  • hHtrA2/Omi
  • mHtrA2/Omi
  • pGEX expression system

ASJC Scopus subject areas

  • Biotechnology

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