N-terminal truncation circumvents proteolytic degradation of the human HtrA2/Omi serine protease in Escherichia coli: rapid purification of a proteolytically active HtrA2/Omi.
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.
Seong
Young-Mo
YM
Graduate School of Biotechnology, Korea University, Seoul 136-701, Republic of Korea.
Park
Hyo-Jin
HJ
Seong
Geun-Hye
GH
Choi
Ju-Youn
JY
Yoon
Sung-Joo Kim
SJ
Min
Byung-Re
BR
Kang
Seongman
S
Rhim
Hyangshuk
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D051379:Mice; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2; C000615915:Htra2 protein, mouse