EXPRESSION, REFOLDING, AND AUTOCATALYTIC PROTEOLYTIC PROCESSING OF THE INTERLEUKIN-1-BETA CONVERTING-ENZYME PRECURSOR

The interleukin-1 beta p-converting enzyme is a heterodimeric cysteine protease that is produced as a 45-kDa precursor, The full-length prec ursor form of the enzyme was expressed in Escherichia coli as insolubl e inclusion bodies. Following solubilization and refolding of the 45-k Da protein, autoproteolytic conversion to a heterodimeric form contain ing 10- and 20-kDa subunits was observed. This enzyme had catalytic ac tivity against both natural (interleukin-1 beta precursor) and synthet ic peptide substrates. The inclusion of a specific inhibitor (SDZ 223- 941) of the converting enzyme in the refolding mixture prevented prote olytic processing to the 10-/20 kDa form. Similarly, refolding under n onreducing conditions also prevented processing. Time course experimen ts showed that the 10-kDa subunit was released from the 45-kDa precurs or before the 20-kDa subunit, implying that the N-terminal portion of the precursor is released last and may play a regulatory role,