Isolation and characterization of biologically active murine interleukin-1 alpha derived from expression of a synthetic gene in Escherichia coli.
A murine interleukin-1 alpha (mIL-1 alpha) gene coding for amino acids 115 to 270 of the precursor protein (Lomedico, P.T., Gubler, U., Hellmann, C.P., Dukovich, M., Giri, J.G., Pan, Y.E., Collier, K., Semionow, R., Chua, A.O. and Mizel, S.B. (1984) Nature 312, 458-462) was chemically synthesized and expressed in Escherichia coli. mIL-1 alpha, in the form of insoluble inclusion bodies, accounted for approx. 30% of total cellular protein produced by the recombinant strain. A simple isolation protocol was developed in which inclusion body material was first solubilized in 3 M guanidine hydrochloride, and the mIL-1 alpha was then simultaneously purified and allowed to fold to its active conformation by dialysis against distilled water. This procedure yielded pure, biologically active mIL-1 alpha with 41% recovery of the mIL-1 alpha present in the guanidine hydrochloride extract. The purified preparation had the expected amino acid composition, a molar absorptivity of 28,200 M-1.cm-1 and a pI of 5.2. No methionyl-mIL-1 alpha was detected by N-terminal sequence analysis, and the endotoxin level was less than 10 pg per micrograms of mIL-1 alpha. The specific biological activity was 3.10(7) units/mg in a co-mitogenic thymocyte proliferation assay. In addition to full-length mIL-1 alpha, the preparation contained N-terminally truncated mIL-1 alpha species (mainly des-4 and des-6 amino acid forms). The truncated species were isolated and found to have the same biological activity as the complete polypeptide. Thus, the active fragment of mIL-1 alpha appears to consist of a proteinase-sensitive N-terminal region which is not essential for activity, and a proteinase-resistant core which harbors the essential determinants of its cytokine function.
Daumy
G O
GO
Department of Molecular Genetics Research, Pfizer Central Research, Groton, CT 06340.
Merenda
J M
JM
McColl
A S
AS
Andrews
G C
GC
Franke
A E
AE
Geoghegan
K F
KF
Otterness
I G
IG
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism*; D005813:Genes, Synthetic; D005822:Genetic Vectors; D002479:Inclusion Bodies / Q000032:analysis; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification; D012995:Solubility
D007375:Interleukin-1; D011498:Protein Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004247:DNA