Baculoviral expression of correctly processed ADAMTS proteins fused with the human IgG-Fc region.
A disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTS) is a novel family of extracellular proteases supposedly involved in inflammation, angiogenesis, development and coagulation. To overexpress the active ADAMTS proteins, we designed a chimeric molecule composed of a catalytic domain of ADAMTS-1 or -4 and the human IgG Fc region in a baculoviral expression system. Both ADAMTS-Fc fusions were produced efficiently in the baculovirus-infected insect cells. The purified fusions underwent cleavage at the predicted furin recognition site. Both ADAMTS-Fc fusions bound to alpha(2)-macroglobulin, further indicating that they were correctly processed with the catalytic activity in this system; however, they failed to digest the peptides derived from the aggrecan sequences known to be clipped by the native enzyme, possibly due to the lack of required multiple interactions existing between the native protease and physiological substrate. In conclusion, the high productivity and facilitated purification of the fusion proteins would offer the source of the biochemical, biophysical or structural studies on the catalytic domain of the ADAMTS proteins.
Sugiura
Takeyuki
T
Discovery Research Laboratory, Tokyo R&D Center, Daiichi Pharmaceutical Co. Ltd., 16-13, Kitakasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan. sugiuy79@daiichipharm.co.jp
D051722:ADAM Proteins; D000818:Animals; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D000072196:Fertilins; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015202:Protein Engineering / Q000379:methods; D011786:Quality Control; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera / Q000201:enzymology* / Q000235:genetics*; D014162:Transfection / Q000379:methods
D007141:Immunoglobulin Fc Fragments; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D051722:ADAM Proteins; C093559:Adam4 protein, mouse; D000072196:Fertilins; D008666:Metalloendopeptidases