Year-end Sale % OFF 
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.
Highlights
Recombinant DNA technology has revolutionized the production and application of pharmaceutical proteins
Yield could be increased to 0.55% of total soluble protein (TSP) by transient expression of human IL-10 fused to an elastin-like polypeptide combined with retention in the endoplasmic reticulum (ER), but biological activity was not confirmed [14]
We show that extensive multimerisation of human IL-10 is the most important factor limiting yield and not protein instability
Summary
Recombinant DNA technology has revolutionized the production and application of pharmaceutical proteins. More attention in optimizing the production of biopharmaceutical proteins is directed to their intrinsic properties that result of post-translational modifications and folding processes Improved insight in these processes may increase the yield of still poorly expressed proteins. Yield could be increased to 0.55% of TSP by transient expression of human IL-10 fused to an elastin-like polypeptide combined with retention in the endoplasmic reticulum (ER), but biological activity was not confirmed [14] From these experiments it was concluded that protein instability is a major bottleneck for human IL-10 production. Domain swapping could be prevented by engineering a stable monomer [15] that regained biological activity through fusion to the Fc portion of IgA, a natural dimer Identification of this expression bottleneck enabled us to increase yield considerably to levels that approach the economic threshold
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
