3D Domain Swapping Causes Extensive Multimerisation of Human Interleukin-10 When Expressed In Planta

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. In this study we show 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 (Fcα), a natural dimer. Stable dimeric forms of IL-10, like Fcα-IL-10, may not only be a better format for improved production, but also a more suitable format for medical applications.

Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants

“…The H58F mutation in the catalytic site of omega-1 to remove RNase activity was introduced by means of overlap extension PCR. The full sequences were cloned into a pHYG expression vector26. Expression vectors (pBIN-PLUS27) for hybrid α1,3-fucosyltransferase IXa (Fut9a) from Tetraodon nigriviridus (puffer fish) and hybrid β1,4-galactosyltransferase (GalT) from Danio rerio (zebrafish) to synthesize LeX structures were used2223.…”

Monomeric IgA can be produced in planta as efficient as IgG, yet receives different N‐glycans

“…Fragments were assembled using standard cloning procedures and ligated into the shuttle vector pRAPa, a pRAP (or pUCAP35S) (van Engelen et al, 1994) derivative modified to include an AsiSI restriction site by introduction of the self-annealed oligo 5 0 -AGCTGGCGATCGCC-3 0 into a HindIII linearized pRAP. The kappa chain expression cassette was digested from pRAPa with AscI and PacI and ligated into the expression vector pHYG (Westerhof et al, 2012). Subsequently, the heavy chain cassettes were combined with the kappa chain expression vector using the restriction sites AscI and AsiSI, of which the latter creates the same overhang as PacI.…”

The N‐glycan on Asn54 affects the atypical N‐glycan composition of plant‐produced interleukin‐22, but does not influence its activity

“…We expressed IL‐22 transiently in N. benthamiana by agroinfiltration and compared its yield to human IL‐10. IL‐10 is structurally similar to IL‐22 and we reported previously that extensive multimerization of IL‐10 is a major expression bottleneck for this cytokine (Westerhof et al ., ). Extensive multimerization of IL‐10 results in the formation of large insoluble granules, but was not observed for IL‐22 in this study.…”