Optimization of Expression and Purification of Recombinant Mouse plac1

Background: Placenta-specific 1 (PLAC1) is one of the recently-discovered Cancer-Testis-Placenta (CTP) antigen with restricted normal tissue and ectopic expression in a wide range of cancer cells from different histological origins. The production of recom-binant human PLAC1 has already been optimized; however, no study has been re-ported so far on the production and purification of mouse plac1. In this study, mouse plac1 expression and purification was optimized in a prokaryotic system and the ef-fects of the generated proteins on inducing humoral responses in mice were investi-gated. Methods: A fusion protein containing full extracellular domain of mouse plac1, im-munostimulatory peptides, tetanus toxin P2P30 and PADRE and KDEL3 signal (main plac1), and the same fragment without immunostimulatory peptides (control plac1) was produced. To optimize production and purification steps, different parameters in-cluding bacterial strain, cultivation temperature, cultivation time, IPTG concentra-tion, culture medium, and also different buffers for purification of the recombinant proteins were tested. After confirming the identity of recombinant plac1 proteins with Western Blotting (WB) and ELISA assays, these proteins were subcutaneously injected in mice with Freund's adjuvant and the anti-plac1 antibody response was detected by ELISA. Results: The optimal expression level of main and control plac1 was obtained in BL21 (DE3) and TB culture medium in the presence of 0.25 mM IPTG after 24 hr of induc-tion at 15C. The buffer containing 2% sarkosyl produced higher yield and purity. Our results showed specific reactivity of anti-human recombinant plac1 polyclonal anti-body with both main and control plac1 recombinant proteins in WB and ELISA analy-sis. Both proteins induced humoral responses in mice; however, anti-plac1 antibody ti-ter was significantly higher in sera of mice immunized with main compared to control plac1. Conclusion: In this study, an optimized protocol for production and purification of mouse plac1 was reported and it was shown that insertion of immunostimulatory pep-tides in gene construct could efficiently enhance humoral immune responses against mouse plac1, which could potentially augment cellular immune responses against plac1 leading to more effective anti-cancer responses.