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, Seong Man Kang, Hyangshuk Rhim

Research output: Contribution to journalArticle

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 1

Fingerprint

Escherichia coli
Purification
Cell death
Degradation
Cell Death
Proteins
Peptide Hydrolases
Apoptosis
Substrates
Omi serine protease
trypsin-like serine protease

Keywords

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

ASJC Scopus subject areas

  • Biochemistry

Cite this

N-terminal truncation circumvents proteolytic degradation of the human HtrA2/Omi serine protease in Escherichia coli : Rapid purification of a proteolytically active HtrA2/Omi. / Seong, Young Mo; Park, Hyo Jin; Seong, Geun Hye; Choi, Ju Youn; Yoon, Sung Joo Kim; Min, Byung Re; Kang, Seong Man; Rhim, Hyangshuk.

In: Protein Expression and Purification, Vol. 33, No. 2, 01.02.2004, p. 200-208.

Research output: Contribution to journalArticle

Seong, Young Mo ; Park, Hyo Jin ; Seong, Geun Hye ; Choi, Ju Youn ; Yoon, Sung Joo Kim ; Min, Byung Re ; Kang, Seong Man ; Rhim, Hyangshuk. / N-terminal truncation circumvents proteolytic degradation of the human HtrA2/Omi serine protease in Escherichia coli : Rapid purification of a proteolytically active HtrA2/Omi. In: Protein Expression and Purification. 2004 ; Vol. 33, No. 2. pp. 200-208.
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AU - Choi, Ju Youn

AU - Yoon, Sung Joo Kim

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AU - Kang, Seong Man

AU - Rhim, Hyangshuk

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AB - 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.

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