Purification and characterization of human recombinant interleukin-1 beta.
A human interleukin-1 (IL-1) beta cDNA was cloned, and the region coding for the mature protein was expressed in Escherichia coli. The 17-kDa biologically active product was purified in 40% yield to apparent homogeneity, without chaotropes, from the soluble fraction of sonicated cell lysates. The recombinant IL-1 beta was characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, and biological assay. Specific biological activity was 4.6 X 10(8) units/mg in a co-mitogenic IL-2 induction assay using cultured EL-4 T-lymphocytes. The molar extinction coefficient was determined to be 10,300 cm-1 M-1 at 280 nm. NH2-terminal sequence analysis revealed that 70% of the product begins with the Ala corresponding to the NH2 terminus of the natural protein, while 30% begins with the following Pro. No initiator Met was observed. Both of the sulfhydryl groups are reactive to Ellman's reagent and to iodoacetamide under nonreducing conditions, indicating that the Cys residues do not form disulfide bonds. S-Carboxamidomethyl-Cys-rIL-1 beta retained biological activity in the IL-2 induction assay. Circular dichroism suggested an extensive beta sheet structure for rIL-1 beta.
Meyers
C A
CA
Johanson
K O
KO
Miles
L M
LM
McDevitt
P J
PJ
Simon
P L
PL
Webb
R L
RL
Chen
M J
MJ
Holskin
B P
BP
Lillquist
J S
JS
Young
P R
PR
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001681:Biological Assay; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D008938:Mitosis; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013438:Sulfhydryl Compounds / Q000032:analysis; D013601:T-Lymphocytes / Q000166:cytology
D000596:Amino Acids; D007375:Interleukin-1; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds