The production and purification for NY-ESO-1 protein were presented by the following steps. First, we assembled a bacterial expression plasmid PET32a (Invitrogen) and NY-ESO-1 (Invitrogen). Then we injected PET32a-NY-ESO-1 into the E. coli strain BL21 (DE3)-bearing to make it recombine, and induced it with IPTG for protein production. Finally we lysed the E. coli using a high-pressure homogenizer (APV 2000, Lubeck, Germany) to attain crude NY-ESO-1 protein (Trx-NY-ESO-1). Here are four steps for the purification of NY-ESO-1 protein, which included Ni-chelating Sepharose affinity chromatography (GE Healthcare, Piscataway, NJ), excision of the Trx-His6-tag, removal of the Trx-His6-tag with second Ni-chelating Sepharose affinity chromatography, and Q-ion-exchange chromatography (GE Healthcare, Piscataway, NJ). The endotoxin level was below standard level.
The preparation of combine vaccine needs the following steps. In short, 20 μg CpG ODN was mixed with 40 μg HH2, and then combined with 125 μg alum, next mixed with 5 μg recombinant NY-ESO-1 protein in PBS with a total volume of 100 μL. The endotoxin levels were approximately 0.02 EU/μg.
The preparation of combine vaccine needs the following steps. In short, 20 μg CpG ODN was mixed with 40 μg HH2, and then combined with 125 μg alum, next mixed with 5 μg recombinant NY-ESO-1 protein in PBS with a total volume of 100 μL. The endotoxin levels were approximately 0.02 EU/μg.