Expression, purification, and functional analysis of the human serine protease HtrA2.
HumHtrA2 or Omi is a recently described member of a novel family of mammalian serine proteases homologous to the Escherichia coli htrA gene product. Although the physiological function of members of this new family is unclear, the current understanding is that as well as being involved with the degradation aberrantly folded proteins during conditions of cellular stress, they may possess a chaperone-like role under normal conditions. In this report we describe the overexpression of humHtrA2 in two heterologous systems comparing the merits of each. We found that molecular analysis of processing events in Sf9 cells allowed us to revisit E. coli expression systems which were initially unsuccessful. Using E. coli we were able to produce milligram amounts of >90% pure recombinant enzyme as determined by SDS-PAGE gels. By means of fluorescently labeled substrates alpha- and beta-casein and zymography, the proteolytic activity of recombinant HumHtrA2 was also demonstrated.
Savopoulos
J W
JW
SmithKline Beecham Pharmaceuticals, New Frontiers Science Park North, Coldharbour Road, Harlow Essex, CM19 5AD, United Kingdom. John_Savapoulos-1@sbphrd.com
Carter
P S
PS
Turconi
S
S
Pettman
G R
GR
Karran
E H
EH
Gray
C W
CW
Ward
R V
RV
Jenkins
O
O
Creasy
C L
CL
D016367:Baculoviridae / Q000235:genetics; D002364:Caseins / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D002364:Caseins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2