Vivaspin 15r centrifugal concentrator

The expression and purification of PAI-1-W175F, PAI-1-stab, Nb42, and Nb64 were performed as previously described [41 (link),42 (link)]. Briefly, the proteins were produced in the Escherichia coli Rosetta^TM 2(DE3)pLysS strain (Merck) as His₆-tagged SUMO fusion proteins in auto-induction ZYP-5052 media [43 (link)]. The cell pellet was resuspended in buffer (50 mM sodium phosphate, 250 mM NaCl, 12.5 mM imidazole, pH 7 (PAI-1) or pH 7.5 (Nbs)). The fusion proteins were purified from the cell lysate using a 5 mL HisTrap HP column (GE Healthcare, Chicago, IL, USA) and treated with SUMO-hydrolase (1:250 mass ratio protein:SUMO hydrolase) to remove the His₆-tagged SUMO domain. Subsequently, this mixture was loaded on a 5 mL HisTrap HP column and the flow through containing the respective non-tagged PAI-1 variant or Nb was collected, dialyzed to ion exchange buffer (20 mM sodium phosphate, pH 7.5), loaded onto a 10 mL HiTrap SP column (GE Healthcare), and eluted by a 0–1 M NaCl gradient. Subsequently, fractions containing PAI-1 or Nb were collected and concentrated using a Vivaspin 15R centrifugal concentrator with a 10 kDa molecular mass cut-off (Sartorius) and further purified by gel filtration on a HiLoad 26/60 Superdex 200 column (GE Healthcare) equilibrated in 20 mM Bis-Tris, 300 mM NaCl, pH 6. All purification steps were conducted on ice or at 4 °C.

F5 isolation was performed according to our previous study (Yap et al., 2015c (link)) where, in brief, cold water extraction was carried out in a mass to volume ratio of 1:20 (g/ml) at 4 °C for 24 h. The extraction mixture was then filtered and freeze dried prior to Sephadex® G-50 fractionation. Proteins from the medium-molecular-weight pooled fraction were then precipitated using 100% saturation ammonium sulfate in a mass-to-volume ratio of 1:30 (g/ml) at 4 °C for an hour and recovered with Vivaspin® 15R Centrifugal Concentrator (Sartorius Stedim Biotech, Göttingen, Germany) with 2 kDa molecular weight cut-off by centrifugation at 6,000×g, 4 °C followed by chromatographic fractionation by RESOURCE™ Q (1 ml) anion exchange column (GE Healthcare, Uppsala, Sweden), pre-equilibrated with start buffer (0.02 M Tris–HCl, pH 8.0). Linear NaCl gradient elution was carried out (0–100% 0.5 M NaCl in the starting buffer) at a flow rate of 1 ml/min, for 45 min. F5 was then collected and analyzed for their protease activity.

To investigate the solubility, antibodies were concentrated with a Vivaspin^® 15R Centrifugal Concentrator with MWCO 10,000 (VS15RH01, Sartorius, Göttigen, Germany) up to the concentration at which visible protein aggregation occurred. The concentrated antibodies were left at RT overnight. Then, the following day, they were centrifuged at 16,000× g for 10 min, and the concentration in the soluble fraction was determined.