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Purification and Characterization of Recombinant Activin Receptor Type IIB Kinase Expressed In Sf9 Cells
Author(s) -
Tam Amy Sze Pui,
Bean Kevin,
Kelleher Kerry,
Marvell Todd,
Ross Cindy,
Kim Richard,
Celeste Tony,
Siegel Marshall,
Kriz Ron,
Somers Will,
Stahl Mark,
Lin Laura
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a498-b
Subject(s) - activin receptor , myostatin , acvr2b , smad , phosphorylation , microbiology and biotechnology , activin type 2 receptors , recombinant dna , receptor , biology , sf9 , transforming growth factor , chemistry , skeletal muscle , tgf beta signaling pathway , endocrinology , gene , biochemistry , spodoptera
Act ivin R eceptor Type IIB (ActRIIB) is a member of the transforming growth factor‐β1 (TGF‐β1) superfamily that has been shown to bind to myostatin/GDF‐8. Upon binding to GDF‐8, ActRIIB receptor recruits the type I receptor ALK4/5, phosphorylates ALK4/5 GS‐rich region and leads to phosphorylation and activation of Smad proteins, which then translocate to the nucleus to regulate gene expression. In mice with dominant negative ActRIIB, they exhibited increase muscle mass and muscle tissue but no phenotypcial abnormality. Disruption of binding between GDF‐8 and ActRIIB receptor by administration of GDF‐8 neutralizing antibody in mice also showed an increase of muscle tissue and muscle strength. These results validate ActRIIB receptor as a good target for small molecule inhibitors for treatment of diseases such as muscular dystrophy. To aide in our structure‐based drug design, we have successfully cloned and expressed numerous constructs of ActRIIB kinase domain using the baculovirus expression system. We were able to achieve ≥95% purity of ActRIIB kinase and biochemical characterizations such as activity and phosphorylation state were used to evaluate the different constructs.