Isolation and characterization of biologically active murine interleukin-6 produced in Escherichia coli.
Interleukin-6 (IL-6) is a multi-functional cytokine produced and secreted by several different cell types, including those of the immune system. A cDNA coding for the mature murine IL-6 (mIL-6), which extends from amino acid (aa) 25 through 211, was cloned into a prokaryotic vector and then expressed in Escherichia coli. The recombinant mIL-6 (remIL-6) was isolated from bacterial inclusion bodies by solubilization in 4 M guanidine hydrochloride followed by gel-filtration chromatography. The protein was refolded to an active conformation by dialysis against 25 mM Na. acetate pH 5.5. A final step of purification and concentration on a cation exchange resin yielded pure and biologically active remIL-6. The purified preparation had the expected aa composition, as confirmed by aa analysis and pI of 7.0-7.1. The biological activity of the recombinant protein was measured in two systems; a proliferation assay employing 7TD1 cells, and a fibrinogen biosynthesis assay employing primary rat hepatocytes. Both assay systems demonstrated that the remIL-6 was active in the range of 10(8) units/mg, which is similar to that estimated for native cytokine. Antibodies raised in rabbits against remIL-6 neutralized the biological activity of both recombinant and native IL-6.
Grenett
H E
HE
Department of Cell Biology, University of Alabama, Birmingham 35294.
Danley
D E
DE
Strick
C A
CA
James
L C
LC
Otterness
I G
IG
Fuentes
N
N
Nesbitt
J E
JE
Fuller
G M
GM
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005340:Fibrinogen / Q000096:biosynthesis; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D007700:Kinetics; D008213:Lymphocyte Activation; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology
D015850:Interleukin-6; D011994:Recombinant Proteins; D005340:Fibrinogen