title: Bacterial production of biologically active canine interleukin-1beta by seamless SUMO tagging and removal.
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.
authors.affiliation: Gene Expression and Protein Biochemistry, GlaxoSmithKline, King of Prussia, PA 19406, USA. Robert.B.Kirkpatrick@gsk.com
authors.forename: Robert B
authors.initials: RB
authors.lastname: Kirkpatrick
authors.forename: Michael
authors.initials: M
authors.lastname: Grooms
authors.forename: Feilan
authors.initials: F
authors.lastname: Wang
authors.forename: Heather
authors.initials: H
authors.lastname: Fenderson
authors.forename: John
authors.initials: J
authors.lastname: Feild
authors.forename: Michael A
authors.initials: MA
authors.lastname: Pratta
authors.forename: Craig
authors.initials: C
authors.lastname: Volker
authors.forename: Gilbert
authors.initials: G
authors.lastname: Scott
authors.forename: Kyung
authors.initials: K
authors.lastname: Johanson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D004926:Escherichia coli / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D053583:Interleukin-1beta / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D053583:Interleukin-1beta; D011994:Recombinant Proteins; D025841:Small Ubiquitin-Related Modifier Proteins