Expression, purification and characterization of murine Dkk1 protein.
Dickkopf-1 (Dkk1) protein is a secreted inhibitor of canonical Wnt signaling and modulates that pathway during embryonic development. It is also implicated in several diseases and hence Dkk1 is a potential target for therapeutic intervention. In the present study 6His-tagged Dkk1 expression and secretion was assessed in five mammalian cell types. Only FreeStyle 293-F cells showed significant Dkk1 protein expression in culture medium. High and stable expression of the Dkk1 protein was obtained from a selected stable FreeStyle 293-F clone 3F8, that grows in suspension in serum-free medium. The 3F8 clone showed a high Dkk1 production level (10mg/L) for up to 2 months of culture. A one step purification procedure resulting in large amounts of highly pure and active Dkk1 protein was developed. Purified Dkk1 binds its receptors LRP5 and LRP6, and is able to dose dependently inhibit canonical Wnt signaling. Recombinant Dkk1 is glycosylated, but this modification is not essential for its biological activity. In summary, an abundant source of pure and functionally active Dkk1 protein is developed that will support the identification of inhibitors such as neutralizing antibodies that could find therapeutic use.
Fleury
Damien
D
Galapagos SASU, Protein Sciences, 102 route de Noisy, 93230 Romainville, Seine Saint-Denis, France.
Gillard
Corinne
C
Lebhar
Helène
H
Vayssière
Béatrice
B
Touitou
Robert
R
Rawadi
Georges
G
Mollat
Patrick
P
D000818:Animals; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000302:isolation & purification / Q000378:metabolism*; D026502:LDL-Receptor Related Proteins / Q000378:metabolism; D060492:Low Density Lipoprotein Receptor-Related Protein-5; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D051379:Mice; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D051153:Wnt Proteins / Q000378:metabolism
C501127:Dkk1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D026502:LDL-Receptor Related Proteins; C555482:LRP5 protein, human; C555475:LRP6 protein, human; D060492:Low Density Lipoprotein Receptor-Related Protein-5; D060488:Low Density Lipoprotein Receptor-Related Protein-6; C555483:Lrp5 protein, mouse; C555476:Lrp6 protein, mouse; D011994:Recombinant Proteins; D051153:Wnt Proteins