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Effects of site-specific mutations on biologic activities of recombinant human IL-6.

J N Snouwaert, K Kariya and D M Fowlkes
J Immunol January 15, 1991, 146 (2) 585-591;
J N Snouwaert
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K Kariya
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D M Fowlkes
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Abstract

To examine structure-activity relationships of human IL-6, we have determined the effects of specific mutations on the biologic activity of a human rIL-6 expressed in bacteria. Three types of mutants were examined: 1) a variant that contains serines in place of the four naturally occurring cysteines; 2) a series of cysteine-containing deletion mutants, each having a single internal 20 amino acid deletion; and 3) a cysteine-free variant containing a single 20 amino acid deletion. The mutants of the second type constitute a set of nonoverlapping, adjacent deletions spanning amino acids 4 through 183 of the 184 amino acids in natural human IL-6. All of the mutants were expressed, along with the full length, cysteine-containing analogue, in Escherichia coli as fusion proteins, joined to beta-galactosidase through a collagen linker. This system allows microgram quantities of the rIL-6 variants to be partially purified from small bacterial cultures without chromatographic or refolding steps. Each of the rIL-6 variants was released from the beta-galactosidase fusion protein with collagenase, and the recovered rIL-6 was quantitated by laser densitometry of Coomassie-stained, SDS polyacrylamide gels. The sp. ac. of each of the rIL-6 variants was determined using four assays: induction of IgM secretion from an EBV transformed human B cell line, induction of fibrinogen secretion from a human hepatoma cell line, induction of fibrinogen secretion from a rat hepatoma cell line, and induction of proliferation of a murine hybridoma cell line. Replacement of cysteines with serines reduced activity relative to cysteine-containing rIL-6 to about 20% in the rat hepatoma assay and about 3% in the mouse hybridoma assay, whereas activity in both of the human cell lines was reduced to less than 0.1%. These data suggest that the murine and rat cell lines are less selective than the human cell lines in their requirements for recognition of biologically active IL-6. Each of the deletions, except that of amino acids 4 through 23, resulted in loss of activity in all four assays. These results suggest that the information necessary for activity is not contained within any one portion of the IL-6 molecule, but rather that multiple segments of the protein are required for each of the biologic activities that we tested.

  • Copyright © 1991 by American Association of Immunologists

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The Journal of Immunology
Vol. 146, Issue 2
15 Jan 1991
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Effects of site-specific mutations on biologic activities of recombinant human IL-6.
J N Snouwaert, K Kariya, D M Fowlkes
The Journal of Immunology January 15, 1991, 146 (2) 585-591;

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Effects of site-specific mutations on biologic activities of recombinant human IL-6.
J N Snouwaert, K Kariya, D M Fowlkes
The Journal of Immunology January 15, 1991, 146 (2) 585-591;
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Print ISSN 0022-1767        Online ISSN 1550-6606