Tarigan Simson, Sekarmila Gita, Tarigan Ronald, Putri Riyandini, Setyawati Damai Ria
Research Organization for Health, National Research and Innovation (BRIN), Cibinong, Indonesia.
School of Veterinary and Medical Sciences, IPB University, Bogor, Indonesia.
PLoS One. 2024 Dec 26;19(12):e0306153. doi: 10.1371/journal.pone.0306153. eCollection 2024.
Developing intranasal vaccines against pandemics and devastating airborne infectious diseases is imperative. The superiority of intranasal vaccines over injectable systemic vaccines is evident, but developing effective intranasal vaccines presents significant challenges. Fusing a protein antigen with the catalytic domain of cholera toxin (CTA1) and the two-domain D of staphylococcal protein A (DD) has significant potential for intranasal vaccines. In this study, we constructed two fusion proteins containing CTA1, tandem repeat linear epitopes of the SARS-CoV-2 spike protein (S14P5 or S21P2), and DD. Structural predictions indicated that each component of the fusion proteins was compatible with its origin. In silico analyses predicted high solubility for both fusion proteins when overexpressed in Escherichia coli. However, contrary to these predictions, the constructs exhibited limited solubility. Lowering the cultivation temperature from 37°C to 18°C did not improve solubility. Inducing expression with IPTG at the early log phase significantly increased soluble CTA1-(S21P2)4-DD but not CTA1-(S14P5)4-DD. Adding non-denaturing detergents (Nonidet P40, Triton X100, or Tween 20) to the extraction buffer significantly enhanced solubility. Despite this, purification experiments yielded low amounts, only 1-2 mg/L of culture, due to substantial losses during the purification stages. These findings highlight the challenges and potential strategies for optimizing soluble expression of CTA1-DD fusion proteins for intranasal vaccines.
开发针对大流行和严重空气传播传染病的鼻内疫苗势在必行。鼻内疫苗相对于注射用全身疫苗的优势显而易见，但开发有效的鼻内疫苗面临重大挑战。将蛋白质抗原与霍乱毒素（CTA1）的催化结构域和葡萄球菌蛋白A的双结构域D（DD）融合，对鼻内疫苗具有巨大潜力。在本研究中，我们构建了两种融合蛋白，它们包含CTA1、严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白的串联重复线性表位（S14P5或S21P2）以及DD。结构预测表明，融合蛋白的每个组分与其来源是兼容的。计算机分析预测，当在大肠杆菌中过表达时，这两种融合蛋白都具有高溶解性。然而，与这些预测相反，构建体的溶解性有限。将培养温度从37°C降至18°C并不能提高溶解性。在对数生长期早期用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，可显著增加可溶性CTA1-(S21P2)4-DD的产量，但不能增加CTA1-(S14P5)4-DD的产量。在提取缓冲液中添加非变性去污剂（NP40、曲拉通X100或吐温20）可显著提高溶解性。尽管如此，由于纯化阶段的大量损失，纯化实验的产量很低，每升培养物仅为1-2毫克。这些发现突出了优化用于鼻内疫苗的CTA1-DD融合蛋白可溶性表达的挑战和潜在策略。
