Bacterial production of biologically active canine interleukin-1beta by seamless SUMO tagging and removal |
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Authors: | Kirkpatrick Robert B Grooms Michael Wang Feilan Fenderson Heather Feild John Pratta Michael A Volker Craig Scott Gilbert Johanson Kyung |
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Institution: | Gene Expression and Protein Biochemistry, GlaxoSmithKline, King of Prussia, PA 19406, USA. Robert.B.Kirkpatrick@gsk.com |
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Abstract: | Interleukin 1beta (IL-1beta) 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-1beta, canine models are relatively refractory to human IL-1beta stimulation. Canine IL-1beta cDNA was cloned in order to produce a fully potent species matched preparation of IL-1beta for use specifically in canine models of OA. Established methods for the production of various orthologous IL-1beta proteins from different species are problematic due to the exquisite sensitivity of the mature IL-1beta 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-1beta from Escherichia coli which was found to be a potent inducer of aggrecanase activity in isolated canine articular chondrocytes. This method combines highly efficient aspects of seamless plasmid engineering, protein purification, and precise tag removal. |
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