Production of a truncated soluble human semicarbazide-sensitive amine oxidase mediated by a GST-fusion protein secreted from HEK293 cells.
Elevated levels of semicarbazide-sensitive amine oxidase (SSAO) activity have been observed in several human conditions such as congestive heart failure, diabetes mellitus, and inflammation. The reactive aldehydes and hydrogen peroxide produced by SSAO have been suggested to contribute to the progression of vascular complications associated with these conditions. In addition, SSAO activity has been shown to be involved in the leukocyte extravasation process at sites of inflammation. To facilitate characterization and development of specific and selective inhibitors of SSAO, we have developed a method for production of recombinant human SSAO. The extracellular region (residues 29-763) of human SSAO was expressed in HEK293 cells in fusion with a mutated Schistosoma japonicum glutathione S-transferase (GST) and secreted to the culture medium. The mutGST-SSAO fusion protein was purified in a single step by glutathione-affinity chromatography followed by site-specific cleavage using a GST-3C protease fusion protein to remove the mutGST fusion partner. A second glutathione-affinity chromatography step was then used to capture both the mutGST fusion partner and the GST-3C protease, resulting in milligram quantities of pure, enzymatically active, and soluble recombinant human SSAO.
Ohman
Johan
J
Biovitrum AB, Research and Development, Berzeliusväg 8, Stockholm, SE-112 76, Sweden.
Jakobsson
Emma
E
Källström
Ulla
U
Elmblad
Annette
A
Ansari
Akbar
A
Kalderén
Christina
C
Robertson
Elinor
E
Danielsson
Eva
E
Gustavsson
Anna-Lena
AL
Varadi
Andrea
A
Ekblom
Jonas
J
Holmgren
Erik
E
Doverskog
Magnus
M
Abrahmsén
Lars
L
Nilsson
Joakim
J
D006631:Amine Oxidase (Copper-Containing) / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D002460:Cell Line; D003920:Diabetes Mellitus / Q000188:drug therapy / Q000201:enzymology; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000627:therapeutic use; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000302:isolation & purification; D006333:Heart Failure / Q000188:drug therapy / Q000201:enzymology; D006801:Humans; D007249:Inflammation / Q000188:drug therapy / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification*; D017384:Sequence Deletion
D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D006631:Amine Oxidase (Copper-Containing); D005982:Glutathione Transferase