Caspase 8: an efficient method for large-scale autoactivation of recombinant procaspase 8 by matrix adsorption and characterization of the active enzyme.
A gene coding for a truncated form of human procaspase 8 has been cloned and expressed in Escherichia coli. This construct contains M(206) through D(479) of human procaspase 8, preceded by an N-terminal polyhistidine tag. The recombinant protein, containing 286 amino acids, was expressed in high yield in the form of inclusion bodies (IB). The IB were solubilized in guanidinium chloride and dialyzed against 50% acetic acid. The solution was mixed with 9 volumes of H(2)O and then rapidly diluted from the acidic medium to one containing 1.0 M Tris, pH 8.0, and 5 mM DTT. SDS-PAGE analysis of the soluble, dilute protein solution (20-30 microgram of protein/ml) showed a single 33-kDa band corresponding to the nonprocessed, inactive procaspase 8. Concentration of the dilute protein to levels as high as 2 mg/ml resulted in only modest (1-10%) autocatalytic conversion to the 19- and 11-kDa polypeptide subunits which are characteristic of the activated enzyme. Further concentration of these protein solutions to a near-dry state on the ultrafiltration membrane, followed by washing of the membrane with buffer, led to extracts containing high yields of enzyme showing a specific activity of 8.43 micromol/min/mg against the chromogenic substrate Ac-IETD-pNA. SDS-PAGE, protein sequencing, and mass spectrometric analysis of these extracts showed complete conversion of the 33-kDa procaspase 8 to the 19- and 11-kDa subunits of activated caspase 8. This method allows for preparation of 100-mg quantities of highly pure and active recombinant human caspase 8. Enzyme activity was shown to be associated with a heterotetrameric complex that is converted to an inactive dimer upon storage.
Koeplinger
K A
KA
Protein Science, Pharmacia & Upjohn, Kalamazoo, Michigan, 49007, USA.
Mildner
A M
AM
Leone
J W
JW
Wheeler
J S
JS
Heinrikson
R L
RL
Tomasselli
A G
AG
D000595:Amino Acid Sequence; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004792:Enzyme Precursors; D011994:Recombinant Proteins; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases