Bacterial production of biologically active canine interleukin-1beta by seamless SUMO tagging and removal.
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.
Kirkpatrick
Robert B
RB
Gene Expression and Protein Biochemistry, GlaxoSmithKline, King of Prussia, PA 19406, USA. Robert.B.Kirkpatrick@gsk.com
Grooms
Michael
M
Wang
Feilan
F
Fenderson
Heather
H
Feild
John
J
Pratta
Michael A
MA
Volker
Craig
C
Scott
Gilbert
G
Johanson
Kyung
K
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*
D018076:DNA, Complementary; D053583:Interleukin-1beta; D011994:Recombinant Proteins; D025841:Small Ubiquitin-Related Modifier Proteins