Hitrap sp ff cation exchange column

All chemicals were from Merck KGaA (Saint Luis, MI, USA). VersaMax Microplate and SoftMax Pro were from Molecular Devices (San Jose, CA, USA). Bradford assay, Mini-PROTEAN Tetra Cell and Mini-Protean Tricine precast gels were from Bio-Rad (Hercules, CA, USA). Plus DNA Ladder and PageRuler Low range unstained protein ladder were from Life Technologies (Carlsbad, CA, USA). Stericup filters were from Millipore (Burlington, MA, USA). HiTrap SP FF cation exchange column and HiTrapOctyl FF hydrophobic interaction column were from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Vivacel 250 membrane filter were from Sartorius (Gottinga, Germany). Ultraflex III MALDI-TOF/TOF instrument and Flex Analysis software were from Bruker Daltonik GmbH (Bremen, Germany).

Purification was performed as described (19 (link)). In brief, these constructs were purified using a HiTrapSP-FF cation exchange column (GE Life Sciences) followed by a MonoQ anion exchange column (GE Life Sciences). Peak fractions were dialyzed into Fhod storage buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 1 mM DTT). The purified constructs were aliquoted, flash frozen using liquid nitrogen, and stored at −80 °C.

The DNA sequence coding the extracellular region of hPD-1 (amino acids 34–150) was cloned into the pET28a vector between NCoI and NdeI sites. Recombinant hPD-1 was expressed in E.coli BL21 (DE3) cells. Cells were cultured in TB medium at 37°C until an optical density at 600 nm of 0.5–0.6 was reached, and then induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 37°C for 5 hours. The cells were pelleted at 4000 rpm for 30 minutes, and the pellets were suspended and lysed in the lysis buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 5% glycerol, 0.5% Triton X-100). Inclusion bodies were recovered by centrifugation (12,000 rpm for 30 minutes), and washed 3 times with 20 mM Tris-HCl, pH 8.0, 2 M urea, 2.5% Triton X-100. The inclusion bodies were finally solubilized in 20 mM Tris-HCl pH 8.0, 8 M urea. The solubilized hPD-1 was refolded by rapid dilution into 50 mM Tris-HCl, pH 8.0, 500 mM L-Arg, 24 mM NaCl, 1 mM KCl, 1 mM EDTA under magnetic stirring for 24 h. The refolding mixture was then concentrated and purified by a HiTrap SP FF cation exchange column (GE Healthcare) and a Superdex 75 gel filtration column (GE Healthcare). The purity of the refolded hPD-1 was evaluated by SDS-PAGE.