Magnehis protein purification system

A total of 20 µl Nickel resin (MagneHis™ Protein Purification System, Promega) was saturated with 6His-Smt3 tagged ZUFSP truncations in 200 µl binding buffer (20 mM TRIS pH 7.5, 150 mM NaCl, 20 mM imidazole and 0.1% NP-40) and incubated for 1 h at 4 °C. All truncations containing the catalytic domain were inactivated by a C360A mutation. The resin was washed three times with binding buffer and afterwards incubated with the twofold molar excess of K63-linked ubiquitin chains for 2 h at 4 °C. The washing steps were repeated and the protein was eluted from the beads by addition of 30 µl Laemmli buffer. The proteins were separated via SDS-PAGE and the subsequent western blots were visualized with α-Smt3 (1:10000; kind gift of J. Dohmen, University of Cologne) or α-ubiquitin P4D1 antibody (1:5000; 3936S; Cell Signaling Technology), respectively.

CHO-K1 cells were maintained as previously described [31] (link) and were grown in T75 culture flasks (Corning, Tewksbury, MA) in a water-jacketed incubator at 37°C, 5% CO₂. Cells were transfected at 80–90% confluency in basal media containing only antibiotic-antimycotic with FuGene HD transfection reagent (Promega, Madison, WI) using a 2:1 reagent to plasmid DNA (µg) ratio. Conditioned media from transfected cells was collected 48–72 hours later and was concentrated using a 3 kDa MWCO Macrosep Advance Centrifugal Device (Pall, Ann Arbor, MI). The heterodimeric hormone and/or individual subunits were subsequently purified using the MagneHIS Protein Purification System (Promega, Madison, WI) following recommended guidelines for purification of secreted proteins. The high imidazole concentration in the elution buffer was reduced by diluting 50-fold in phosphate buffered saline containing 0.02% sodium azide. This larger volume containing the purified heterodimeric hormone or individual subunits was concentrated using a centrifugal concentrating device as noted above.

Escherichia coli MC1061 transformed with pBAD:Gp2 was grown as described above and the time points at which samples (50 ml) were taken are indicated in the figures. The MagneHis™ Protein Purification System (Promega) was used for pull-down assays according to manufacturer's instructions. Briefly, the bacterial pellet was resuspended in 3 ml of Tris Buffered Saline (TBS) and lysed by sonication using a Sonics Vibracell sonicator (settings: amplitude 40%, 5 s on, 5 s off pulse for 5 min). The lysate was then mixed with 100 μl of resin beads and incubated for 1 h at 4°C. The beads were washed with 5× resin bed volume of TBS. To elute bound proteins from the beads, 100 μl of 2× SDS sample buffer (0.125 M Tris–HCl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 10% (v/v), 2-mercaptoethanol, 0.004% (w/v) bromophenol blue) was added and the beads were boiled for 1 min at 100°C. Ten microliters of the eluted sample was loaded on a 4–20% gradient SDS-polyacrylamide electrophoresis (PAGE) gel and proteins of interest were detected by western blotting (see below).

Mouse VPCs were isolated from the outgrowth of aortic adventitial tissues as described previously.^{13 (link)} Sca-1+ VPCs were treated with 25 ng/mL of Dkk3 for 3 hours. The lysate was precleared by using the control agarose resin column. The eluted immune complexes and input samples were separated on a 4% to 12% Bis-Tris gel, and the immunoblot was probed with Dkk3 antibody. His-tag pull-down assay was performed according to the instructions provided in MagneHis Protein Purification System (Promega). Receptor affinity binding assay was done as described previously.^{22 (link)}

The PirA^VP plasmid (pET21b PirA^VP) and PirB^VP plasmid (pET21b PirB^VP), obtained from Taiwan, were transformed into E. coli Rosetta (DE3) competent cells and the expression of the recombinant, C-terminal His₆-tagged PirA^VP and PirB^VP proteins, respectively, were induced by the addition of 0.25 µM of iso-propyl thiogalactoside (IPTG) [3 (link)]. After expression had proceeded at 16 °C for 16 h, the recombinant proteins were purified with the magneHis™ protein purification system (Promega Corporation, WI, USA). The purified recombinant PirA^VP and PirB^VP toxins were dialyzed in phosphate buffer solution (PBS) (Honeywell, Grauwmeer, Belgium) and concentrated with amicon^® ultra-15 centrifugal filters (Merck Millipore, Overijse, Belgium).