Bacterial production of biologically active canine interleukin-1β by seamless SUMO tagging and removal

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Abstract

Interleukin 1β (IL-1β) is a potent stimulator of extracellular matrix degradation in models of osteoarthritis (OA). In contrast to bovine explant models which effectively respond to recombinant human IL-1β, canine models are relatively refractory to human IL-1β stimulation. Canine IL-1β cDNA was cloned in order to produce a fully potent species matched preparation of IL-1β for use specifically in canine models of OA. Established methods for the production of various orthologous IL-1β proteins from different species are problematic due to the exquisite sensitivity of the mature IL-1β product to N-terminal variations and the intrinsic technical challenges associated with producing an unmodified product. We have applied a seamless method of SUMO tagging and removal in order to produce a homogeneous unmodified preparation of canine IL-1β from Escherichia coli which was found to be a potent inducer of aggrecanase activity in isolated canine articular chondroctyes. This method combines highly efficient aspects of seamless plasmid engineering, protein purification, and precise tag removal.

Section snippets

Reagents

IR800 labeled in house anti-His6 mAb was prepared following the protocol provided by manufacturer, Li-COR. S. cerevisiae genomic DNA was from Promega, G32101. KOD hot start DNA polymerase and expression plasmids pET16b and pET22b were obtained from Novagen (Madison, WI). Pecast Bis–Tris NuPAGE 4–20% SDS–PAGEs, pre-stained molecular weight markers, pENTR/SD-topo vector, pCRII/topo LR and BP clonase and competent BL21(DE3) cells were from Invitrogen (Carlsbad, CA). Quickchange™ site-directed

Results

In order to study changes associated with cartilage destruction in canine experimental osteoarthritis, we have examined IL-1-stimulated cartilage destruction in cartilage explants derived from normal animals. Unlike bovine or human chondrocytes which are stimulated by human IL-1β to secrete matrix degrading enzymes including aggrecanases and MMP13, we found canine cartilage explants and isolated chondrocytes to be poorly responsive to hIL-1β treatments. Therefore, in order to enable experiments

Discussion

We have produced active mature canine IL-1β for use in canine articular models of cartilage degradation. Since human IL-1β is incapable of stimulating cartilage degradation in the canine cartilage explants, and this was associated with a reduced capacity to induce aggrecanase activity from isolated canine chondrocytes, the availability of canine IL-1β will be particularly important in the development of canine models of cartilage degradation. IL-1β is synthesized as an inactive precursor in the

Acknowledgments

We thank Elizabeth Thomas, Kimberly Allen, Wendy Halsey, Ashley Hughes, and Ganesh Sathe; Department of Discovery and Pipeline Genetics, for exceptional DNA sequencing support.

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