Purification of interleukin-1 beta converting enzyme, the protease that cleaves the interleukin-1 beta precursor.
We have purified the IL-1 beta converting enzyme from the THP-1 cell line using standard chromatographic techniques and obtained the N-terminal amino acid sequence of this novel protein. After stimulation of THP-1 cells with lipopolysaccharide, hydroxyurea, and silica, the protease was solubilized by multiple freeze/thawing. The protein was purified by ion-exchange chromatography, affinity chromatography on blue agarose, gel filtration, and chromatofocusing. The molecular weight of the protein is approximately 22,000 Da and the pI is between 7.1 and 6.8. The overall yield for this procedure was 16% of the activity found in the initial cell lysates. An antiserum raised against a peptide based on the N-terminus was used to precipitate the protease, confirming our identification of the 22,000-Da protein as the IL-1 beta converting enzyme.
Kronheim
S R
SR
Protein Chemistry Department, Immunex Corporation, Seattle, Washington 98101.
Mumma
A
A
Greenstreet
T
T
Glackin
P J
PJ
Van Ness
K
K
March
C J
CJ
Black
R A
RA
D000595:Amino Acid Sequence; D015153:Blotting, Western; D020170:Caspase 1; D002460:Cell Line; D002845:Chromatography; D006918:Hydroxyurea / Q000494:pharmacology; D007163:Immunosorbent Techniques; D007375:Interleukin-1 / Q000378:metabolism*; D007526:Isoelectric Point; D008070:Lipopolysaccharides; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011498:Protein Precursors / Q000378:metabolism*; D012822:Silicon Dioxide / Q000494:pharmacology
D007375:Interleukin-1; D008070:Lipopolysaccharides; D011498:Protein Precursors; C043830:interleukin 1 precursor; D012822:Silicon Dioxide; D020170:Caspase 1; D008666:Metalloendopeptidases; D006918:Hydroxyurea