Millex hv filter unit

The PspA gene was amplified by polymerase chain reaction (PCR) and cloned into pET16b plasmid (Novagen, Darmstadt, Germany), as previously described, to yield pET16b-PspA plasmid [17 (link)]. To obtain PspA recombinant proteins, the plasmids were transformed into E. coli strain BL21 (DE3) (Novagen). Protein production was induced by adding isopropyl-β-D-thiogalactopyranoside (Nacalai Tesque, Kyoto, Japan). The culture pellets were sonicated for 1 min three times in buffer A (10 mM Tris-HCl [pH 8.0], 400 mM NaCl, 5 mM MgCl₂, 0.1 mM PMSF, 1 mM 2-mercaptethanol, and 10% glycerol). After centrifugation of the mixture at 4 °C and 17,800× g for 15 min, the supernatants were filtered through a 0.45 µm Millex-HV filter unit (Merck Millipore, Burlington, MA, USA) and loaded into HiTrap HP columns (GE Healthcare, Pittsburgh, PA, USA). PspA was eluted with buffer A containing 100 to 500 mM imidazole. The eluted protein was loaded into a PD-10 column (GE Healthcare) for exchange with PBS (Nacalai Tesque). The concentration of purified protein was measured by using a BCA protein assay kit (Pierce Chemical, Rockford, IL, USA). The purity of the eluted protein was confirmed in a NuPAGE electrophoresis system (Life Technologies, Carlsbad, CA, USA) followed by staining with Coomassie brilliant blue (Nacalai Tesque).