Expression, refolding, and autocatalytic proteolytic processing of the interleukin-1 beta-converting enzyme precursor.
The interleukin-1 beta-converting enzyme is a heterodimeric cysteine protease that is produced as a 45-kDa precursor. The full-length precursor form of the enzyme was expressed in Escherichia coli as insoluble inclusion bodies. Following solubilization and refolding of the 45-kDa protein, autoproteolytic conversion to a heterodimeric form containing 10- and 20-kDa subunits was observed. This enzyme had catalytic activity against both natural (interleukin-1 beta precursor) and synthetic peptide substrates. The inclusion of a specific inhibitor (SDZ 223-941) of the converting enzyme in the refolding mixture prevented proteolytic processing to the 10-/20-kDa form. Similarly, refolding under nonreducing conditions also prevented processing. Time course experiments showed that the 10-kDa subunit was released from the 45-kDa precursor before the 20-kDa subunit, implying that the N-terminal portion of the precursor is released last and may play a regulatory role.
Ramage
P
P
Department of Biotechnology, Sandoz Pharma Ltd., Basel, Switzerland.
Cheneval
D
D
Chvei
M
M
Graff
P
P
Hemmig
R
R
Heng
R
R
Kocher
H P
HP
Mackenzie
A
A
Memmert
K
K
Revesz
L
L
D000595:Amino Acid Sequence; D020170:Caspase 1; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D017510:Protein Folding
D004792:Enzyme Precursors; D003546:Cysteine Endopeptidases; D020170:Caspase 1