Resolving 3D-domain swapping of human IL-10 prevents extensive multimerisation and increases yield

L.B. Westerhof, R.H.P. Wilbers, J. Roosien, J. Bakker, A. Schots

Research output: Chapter in Book/Report/Conference proceedingAbstractAcademic

Abstract

Heterologous expression platforms of biopharmaceutical proteins have been significantly improved over the last decade. Further improvement can be established by examining the intrinsic properties of proteins. Interleukin-10 (IL-10) is an anti-inflammatory cytokine with a short half-life that plays an important role in re-establishing immune homeostasis. This homodimeric protein of 36 kDa has significant therapeutic potential to treat inflammatory and autoimmune diseases. We have shown that the major production bottleneck of human IL-10 is not protein instability as previously suggested, but extensive multimerisation due to its intrinsic 3D domain swapping characteristic. Extensive multimerisation of human IL-10 could be visualised as granules in planta. On the other hand, mouse IL-10 hardly multimerised, which could be largely attributed to its glycosylation. By introducing a short glycine-serine-linker between the fourth and fifth alpha helix of human IL-10 a stable monomeric form of IL-10 (hIL-10mono) was created that no longer multimerised and increased yield up to 20-fold. However, hIL-10mono no longer had the ability to reduce pro-inflammatory cytokine secretion from lipopolysaccharide-stimulated macrophages. Forcing dimerisation restored biological activity. This was achieved by fusing human IL-10mono to the C-terminal end of constant domains 2 and 3 of human immunoglobulin A (Fca), a natural dimer. Stable dimeric forms of IL-10, like Fca-IL-10, may not only be a better format for improved production, but also a more suitable format for medical applications.
Original languageEnglish
Title of host publicationMolecular farming: plants as production platform for high value proteins
Place of PublicationWarsaw
Pages27-27
Publication statusPublished - 2012
EventCost FA0804 Molecular farming: plants as production platform for high value proteins, Warsaw, 5-7 September 2012 -
Duration: 5 Sep 20127 Sep 2012

Conference

ConferenceCost FA0804 Molecular farming: plants as production platform for high value proteins, Warsaw, 5-7 September 2012
Period5/09/127/09/12

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