Recombinant Production and Purification of ALK1 Extracellular Domain

Human ALK1 cDNA (NM_000020) encoding amino acids 22-118 was cloned into pET39b (70090, Novagen) between NcoI and NotI sites to create a fusion protein DsbA-(His)₆-ALK1 ECD. A TEV (Tobacco Etch Virus nuclear inclusion A endopeptidase) protease cleavage site was introduced at the N-terminus of the ALK1 ECD to facilitate release of the untagged ECD. The construct was confirmed by DNA sequencing and transformed into bacterial strain Rosetta DE3 (70954, Novagen) for protein expression. Cells were grown to mid-log phase followed by isopropyl β-D-thiogalactopyranoside induction and further incubation at 22 °C overnight. ALK1 ECD was purified from periplasmic fractions following the method described previously for the BMPRII ECD^{5 (link)}. Briefly, total periplasmic proteins were extracted following the pET System Manual (Novagen) and His-tagged fusion proteins were purified on a 5 ml nickel-nitrilotriacetic acid column (GE Healthcare). Fractions containing the fusion protein were pooled, dialysed into Tris Buffered Saline, and incubated overnight with His-tagged TEV protease. The mixture was passed through a pre-charged nickel-nitrilotriacetic acid column to remove His-tagged DsbA and TEV protease. ALK1 ECD, which was in the flow-through and wash solution, was concentrated and further purified by gel filtration on a Superdex 75 column.