Overexpression and structure--function analysis of a bioengineered IL-2/IL-6 chimeric lymphokine.
A synthetic chimeric IL-2/IL-6 gene was synthesized to engineer a bifunctional lymphokine which was overproduced in Escherichia coli. Following denaturation of the inclusion bodies in 6 M guanidine and refolding and reoxidation in the presence of a redox system, the fusion protein (rIL-2/IL-6) was purified to homogeneity and shown to react with both monospecific anti-IL-2 and anti-IL-6 antisera. A collagen-like spacer was introduced between the two cytokine moieties to generate IL-2 and IL-6 molecules upon collagenase digestion. After cleavage, the two subunits, purified in a single-step procedure, were found to be correctly reoxidized and functionally as active as their native counterparts. Circular dichroism studies of rIL-2/IL-6 revealed that both cytokine subunits refolded independently and exhibited the alpha-helical structures characteristic of the corresponding wild-type lymphokines. The chimera displayed full IL-2 activity in the CTLL-2 cell proliferation assay. It also retained the IL-6 property to enhance IgM synthesis in SKW6.4 cells, induce the proliferation of B-cell hybridomas and stimulate the production of fibrinogen in hepatocytes. Because IL-2 amplifies the cellular immune response and IL-6 up-regulates the humoral response, this bifunctional lymphokine represents a potentially useful therapeutic adduct and may serve as an immunomodulator to enhance the host's response to vaccination.
Rock
F
F
Department of Biochemistry, University of Toronto, Ontario, Canada.
Everett
M
M
Klein
M
M
D000595:Amino Acid Sequence; D000917:Antibody Formation / Q000187:drug effects*; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004926:Escherichia coli; D005340:Fibrinogen / Q000096:biosynthesis; D007111:Immunity, Cellular / Q000187:drug effects*; D007075:Immunoglobulin M / Q000096:biosynthesis; D007376:Interleukin-2 / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D015850:Interleukin-6 / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects; D008213:Lymphocyte Activation / Q000187:drug effects; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000494:pharmacology
D007075:Immunoglobulin M; D007376:Interleukin-2; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D005340:Fibrinogen