Bacterial production of biologically active canine interleukin-1β by seamless SUMO tagging and 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.
References (29)
- et al.
Crystal structure of recombinant human interleukin-1β at 2.0A resolution
J. Mol. Biol.
(1989) - et al.
A sequence-based map of the nine genes of the human interleukin-1 cluster
Genomics
(2002) - et al.
Cloning and expression of an interleukin-1 beta precursor and its conversion to interleukin-1 beta
FEBS Lett.
(1989) - et al.
Purification and characterization of human recombinant interleukin-1 beta
J. Biol. Chem.
(1987) - et al.
Temperature, media, and point of induction affect the N-terminal processing of interleukin-1 beta
Protein Expr. Purif.
(2005) - et al.
Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element
Gene
(1997) - et al.
A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa
Protein Expr. Purif.
(2003) - et al.
Expression and purification of SARS coronavirus proteins using SUMO-fusions
Protein Expr. Purif.
(2005) - et al.
SUMO fusion technology for difficult-to-express proteins
Protein Expr. Purif.
(2005) - et al.
Development and characterization of a highly specific and sensitive sandwich ELISA for detection of aggrecanase-generated aggrecan fragments
Osteoarthritis Cart.
(2006)