Malaria and Vector Research Group, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran; , Department of Parasitology and Mycology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.
Malaria and Vector Research Group, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Protein Expr Purif. 2022 Oct;198:106126. doi: 10.1016/j.pep.2022.106126. Epub 2022 May 31.
Generative cell specific 1 (GCS1) or Hapless2 (Hap2) is a main transmission-blocking vaccine (TBV) candidate against malaria. Experience has shown that this protein is difficult to express in heterologous hosts. In a study, Plasmodium falciparum GCS1 (PfGCS1) could be expressed in fusion with Glutathione S Transferase (GST). Since the large fusions could influence the immunogenicity of the recombinant antigens, in the current study, we tried to express PfGCS1 protein without large fusion tags with an appropriate yield and purity in E. coli. To this end, pfgcs1 gene was codon-optimized and cloned in pET23a plasmid. The expression was evaluated in different E. coli hosts [E. coli BL21(DE3), E. coli BL21(DE3) pLysS, E. coli Rosetta(DE3), and E. coli Rosettagami(DE3)] and media cultures. In addition, the effect of post-induction times, inducer concentration, temperature, and supplementation of glucose and ethanol to culture media were evaluated. The obtained results revealed that rPfGCS1 protein was expressed in all examined E. coli hosts and media cultures with different yields, with the best yield in E. coli BL21(DE3), and E. coli Rosetta(DE3) hosts in TB medium, 16 h post-induction. The expression of rPfGCS1 was confirmed by western blotting using anti-His antibodies. Expression in low temperature at 20 °C and addition of glucose and ethanol to TB media could improve the expression of rPfGCS1. We could express and purify rPfGCS1 without a large fusion protein with an appropriate yield and purity in E. coli Rosetta(DE3). We will evaluate this antigen as TBV candidate against P. falciparum transmission in the future.
生殖细胞特异性 1（GCS1）或不幸 2（Hap2）是一种主要的疟疾传播阻断疫苗（TBV）候选物。经验表明，这种蛋白质很难在异源宿主中表达。在一项研究中，疟原虫 GCS1（PfGCS1）可以与谷胱甘肽 S 转移酶（GST）融合表达。由于大融合标签可能会影响重组抗原的免疫原性，因此在本研究中，我们试图在大肠杆菌中以适当的产量和纯度表达无大融合标签的 PfGCS1 蛋白。为此，对 pfgcs1 基因进行了密码子优化，并克隆到 pET23a 质粒中。在不同的大肠杆菌宿主[大肠杆菌 BL21（DE3）、大肠杆菌 BL21（DE3）pLysS、大肠杆菌 Rosetta（DE3）和大肠杆菌 Rosettagami（DE3）]和培养基中评估了表达情况。此外，还评估了诱导后时间、诱导剂浓度、温度以及在培养基中添加葡萄糖和乙醇的影响。结果表明，rPfGCS1 蛋白在所有检测的大肠杆菌宿主和培养基中均有不同的产量表达，在 TB 培养基中，大肠杆菌 BL21（DE3）和大肠杆菌 Rosetta（DE3）宿主的产量最高，诱导后 16 小时。用抗 His 抗体进行 Western blot 验证了 rPfGCS1 的表达。在 20°C 的低温下表达并在 TB 培养基中添加葡萄糖和乙醇可以提高 rPfGCS1 的表达。我们可以在大肠杆菌 Rosetta（DE3）中表达和纯化无大融合蛋白的 rPfGCS1，产量和纯度适当。我们将在未来评估这种抗原作为疟原虫传播阻断疫苗候选物。
