Overexpression and structure-function analysis of a bioengineered IL-2/IL-6 chimeric lymphokine |
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Authors: | Rock, Fernando Everett, Margaret Klein, Michel |
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Affiliation: | 1Department of Biochemistry Willowdale, Ontario M2R 3T4, Canada 2Department of Immunology, University of Toronto Toronto, Ontario M5S 1A8 3Connaught Centre for Biotechnology Research Willowdale, Ontario M2R 3T4, Canada |
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Abstract: | A synthetic chimeric IL-2/IL-6 gene was synthesized to engineera bifunctional lymphokine which was overproduced in Escherichiacoli. Following denaturation of the inclusion bodies in 6 Mguanidine and refolding and reoxidation in the presence of aredox system, the fusion protein (rIL-2/IL-6) was purified tohomogeneity and shown to react with both monospecific anti-IL-2and anti-IL-6 antisera. A collagen-like spacer was introducedbetween the two cytokine moieties to generate IL-2 and IL-6molecules upon collagenase digestion. After cleavage, the twosubunits, purified in a single-step procedure, were found tobe correctly reoxidized and functionally as active as theirnative counterparts. Circular dichroism studies of rIL-2/IL-6revealed that both cytokine subunits refolded independentlyand exhibited the a-helical structures characteristic of thecorresponding wild-type lymphokines. The chimera displayed fullIL-2 activity in the CTLL-2 cell proliferation assay. It alsoretained the IL-6 property to enhance lgM synthesis in SKW6.4cells, induce the proliferation of B-cell hybridomas and stimulatethe production of fibrinogen in hepatocytes. Because IL-2 amplifiesthe cellular immune response and IL-6 up-regulates the humoralresponse, this bifunctional lyinphokine represents a potentiallyuseful therapeutic adduct and may serve as an immunomodulatorto enhance the host's response to vaccination. |
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Keywords: | chimera/ gene/ IL-2/ IL-6/ interleukins |
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