Purification and characterization of human recombinant precursor interleukin 1 beta.
We have purified the 31-kDa precursor of human interleukin 1 beta (proIL1 beta) from recombinant Escherichia coli expressing the protein. The recombinant precursor was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, Western blot, and for biological and receptor binding activity. The protein migrates at the expected molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration columns. The specific activity of the recombinant precursor is less than 10(2) units/mg in the EL4 thymoma assay compared with 5 x 10(8) units/mg for the recombinant 17-kDa mature protein. The inactivity of the precursor is attributable to the inability of the protein to bind the IL1 receptor on EL4 cells as shown by receptor competition studies using 125I-labeled 17-kDa IL1 beta. Inactivity of the IL1 beta precursor is not due to degradation of the protein in either the bioactivity or receptor binding assays. The inactive IL1 beta precursor is converted to an active form following proteolysis with chymotrypsin which generates a carboxyl-terminal fragment of 17 kDa that is 6 orders of magnitude more active than the starting IL1 beta precursor. Removal of the first 114 amino acids from proIL1 beta generates a fully active molecule. In contrast, removal of the first 77 amino acids by treatment with trypsin only partially restores activity. The resultant 22-kDa protein exhibits a 600-fold increase in both biological and receptor binding activity, demonstrating a direct correlation between the ability of sequences within the pro-region to inhibit biological activity and inhibit binding to the IL1 receptor. Far-UV circular dichroism spectroscopy indicates that proIL1 beta is similar in secondary structure to mature IL1 beta; both proteins are nonhelical beta sheet proteins.
Hazuda
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Department of Molecular Genetics, Smith Kline and French Laboratories, King of Prussia, Pennsylvania 19406-0930.
Webb
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RL
Simon
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Young
P
P
D002942:Circular Dichroism; D006801:Humans; D007375:Interleukin-1 / Q000302:isolation & purification*; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D011498:Protein Precursors / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification
D007375:Interleukin-1; D011498:Protein Precursors; D011994:Recombinant Proteins; C043830:interleukin 1 precursor; D010447:Peptide Hydrolases