Optimization of Expression and Purification of Recombinant Mouse plac1

Shaghayegh  Rahdan; Seyed Alireza  Razavi; Mahboobeh  Nazari; Sorour Shojaeian; Fazel  Shokri; Mohammad Mehdi  Amiri; Amin  Ramezani; Amir-Hassan  Zarnani

doi:10.18502/ajmb.v14i1.8171

Shaghayegh Rahdan Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Seyed Alireza Razavi Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Mahboobeh Nazari Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
Sorour Shojaeian Department of Biochemistry, School of Medical Sciences, Alborz University of Medical Sciences, Karaj, Iran
Fazel Shokri Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Mohammad Mehdi Amiri Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Amin Ramezani Institute for Cancer Research, Shiraz University of Medical Sciences, Shiraz, Iran
Amir-Hassan Zarnani Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

DOI: https://doi.org/10.18502/ajmb.v14i1.8171

Keywords: Expression, Mouse plac1, Optimization, Purification, Recombinant proteins

Abstract

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 recombinant human PLAC1 has already been optimized; however, no study has been reported 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 effects of the generated proteins on inducing humoral responses in mice were investigated.

Methods: A fusion protein containing full extracellular domain of mouse plac1, immunostimulatory 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 including bacterial strain, cultivation temperature, cultivation time, IPTG concentration, 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 induction at 15°C. The buffer containing 2% sarkosyl produced higher yield and purity. Our results showed specific reactivity of anti-human recombinant plac1 polyclonal antibody with both main and control plac1 recombinant proteins in WB and ELISA analysis. Both proteins induced humoral responses in mice; however, anti-plac1 antibody titer 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 peptides 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.