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. Author manuscript; available in PMC: 2022 Aug 6.
Published in final edited form as: Circ Res. 2021 Jun 28;129(4):458–470. doi: 10.1161/CIRCRESAHA.121.319163

Figure 1: Full-length mouse ADAMTS7 purification and in vitro TSP1 cleavage.

Figure 1:

A, ADAMTS7 protein domains and locations of the glutamate to glutamine (EQ) catalytic mutant and serine to alanine (S3A) substitutions to prevent proteoglycan (PG) attachment. Abbreviated ADAMTS7 domains: signal peptide (SP), disintegrin (Dis), thrombospondin repeats (T), cysteine-rich (Cys-rich), protease and lacunin (PL). B, expression of full-length (FL) mouse and human ADAMTS7 3xFlag proteins in the whole cell lysate and secreted in the media under reducing conditions, detected by western blot using the M2-HRP antibody. High molecular weight proteoglycan (PG) species are marked in orange. A longer exposure of the conditioned media was required to visualize the secreted human ADAMTS7 proteins. C, full-length ADAMTS7 two step purification workflow used to purify WT S3A active enzyme and EQ S3A negative control protein. D, Thrombospondin1 in vitro cleavage by purified full-length mouse ADAMTS7 WT protein (* indicates co-purified auto-cleavage band). Western blots under reducing conditions to resolve proteolytic TSP1 bands generated by active ADAMTS7 WT purified enzyme. Presence of the E373Q catalytic mutation in the purified full-length mouse ADAMTS7 EQ protein ablated the catalytic activity.