Rat interleukin-10: production and characterisation of biologically active protein in a recombinant bacterial expression system.
Interleukin-10 is an anti-inflammatory Th1 immunosuppressive cytokine, the active form of which is a non-covalent homodimer, and which exhibits species-specificity both with respect to structure and biological activity. The rat homologue of IL-10 shares 73% identity with human IL-10 at the amino-acid sequence level, and has, in addition to the two disulphide bonds present in human IL-10, a fifth, unpaired cysteine (cys-149). Preparation of rat IL-10 by bacterial expression followed by solubilisation and refolding in a glutathione redox system, results in a molecule in which cys-149 is almost entirely oxidised, existing either as disulphide dimer or as a mixed disulphide with glutathione, and which has less than 1% of the activity of the native (cys-149-SH) form of the molecule. Site directed mutagenesis of rat IL-10 to replace cys-149 with tyrosine produces a molecule which readily adopts the active conformation upon solubilisation and refolding, and which is recoverable in good yield from bacterial expression systems. Comparison of the biological activities of rat IL-10tyr149 and commercial rat IL-10 preparations confirms that the activity of native-sequence rat IL-10 is either reduced or absent. It is proposed therefore that the biosynthetic analogue rat IL-10tyr149 is a more useful molecule to investigate the biological actions of IL-10 in the rat.
Division of Endocrinology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom.
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Ball
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Vigues
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Gee
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Poole
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Bristow
D000818:Animals; D001419:Bacteria / Q000235:genetics*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D016753:Interleukin-10 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D016753:Interleukin-10; D004247:DNA