Nickel nitrilotriacetic acid column

E. coli BL21 (DE3) was transformed with pEtCsMAP34, pEtCsMASP1, and pET32a (which expresses the Trx tag). The transformants were cultured in LB medium at 37 °C to mid-log phase, and the expression of rCsMAP34, rCsMASP1, and rTrx was induced by adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mM. After growth at 16 °C for an additional 16 h, the cells were harvested by centrifugation, and recombinant proteins were purified using nickel-nitrilotriacetic acid columns (GE Healthcare, Piscataway, USA), as recommended by the manufacturer. The purified proteins were reconstituted as described previously49 (link). The reconstituted proteins were treated with Triton X-114 to remove endotoxin as reported previously50 (link). The proteins were dialyzed for 24 h against PBS and concentrated using PEG20000 (Solarbio, Beijing, China). The concentrations of the purified proteins were determined using the Bradford method with bovine serum albumin as a standard. Mouse antibodies against rCsMAP34, rCsMASP1, and rTrx were prepared as described previously51 (link). The antibodies were purified using rProtein G Beads (Solarbio, Beijing, China). The specificity and titer of the serum antibodies were determined by Western immunoblot and enzyme-linked immunosorbent assay (ELISA) as reported previously52 (link).

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.

The recombinant enzymes were purified as described previously in our laboratory (Huang et al., 2015 (link)). Briefly, the E. coli BL21(DE3) cells were induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) overnight at 16°C and collected for protein purification after sonication on ice. Then the proteins were collected by washing and eluting with 80 and 400 mM imidazole through a nickel nitrilotriacetic acid column (GE Healthcare, US), respectively. The collected active protein fractions were pooled and dialyzed against 20 mM Tris-HCl (pH 7.4) and stored at –80°C until use (less than 2 weeks).

Human BMPR2 (NM_001204) ECD, containing residues 27–150, was cloned into pET39b (Novagen) between NcoI and NotI sites to generate a construct expressing DsbA-(His)₆-BMPR2 ECD fusion protein. A TEV protease (Tobacco Etch Virus nuclear inclusion A endopeptidase) cleavage site was introduced at the N terminus of BMPR2 ECD to facilitate the cleavage of the fusion protein. The insert was confirmed by DNA sequencing. The plasmid was transformed into bacterial strain Rosetta DE3 for protein expression. In brief, cells were grown at 37 °C until mid-log phase followed by isopropyl-β-d-thiogalactoside induction at 25 °C overnight. Total proteins in the periplasmic compartment were extracted following the pET System Manual (Novagen) and applied to a 5 ml of nickel-nitrilotriacetic acid column (GE Healthcare). Fractions containing the fusion protein were pooled, dialyzed into TBS, and incubated with His-tagged TEV protease overnight before being loaded again onto a precharged nickel-nitrilotriacetic acid column to remove the DsbA tag and TEV protease. BMPR-II ECD, which was in the flowthrough of the column, was concentrated and further purified on a S75 gel filtration chromatography. The final BMPR-II is over 99% pure on an SDS-PAGE (data not shown).