Queensland Agricultural Biotechnology Facility, Agri-Science Queensland, Queensland, Australia.
Microb Cell Fact. 2011 Jul 26;10:57. doi: 10.1186/1475-2859-10-57.
Protein expression in Escherichia coli may result in the recombinant protein being expressed as insoluble inclusion bodies. In addition, proteins purified from E. coli contain endotoxins which need to be removed for in vivo applications. The structural protein, E2, from Bovine Viral Diarrhoea Virus (BVDV) is a major immunogenic determinant, and is an ideal candidate as a subunit vaccine. The E2 protein contains 17 cysteine residues creating difficulties in E. coli expression. In this report we outline a procedure for successfully producing soluble and endotoxin-free BVDV E2 protein from inclusion bodies (IB).
The expression of a truncated form of BVDV-E2 protein (E2-T1) in E. coli resulted in predominantly aggregated insoluble IB. Solubilisation of E2-T1 with high purity and stability from IB aggregates was achieved using a strong reducing buffer containing 100 mM Dithiothreitol. Refolding by dialysis into 50 mM Tris (pH 7.0) containing 0.2% Igepal CA630 resulted in a soluble but aggregated protein solution. The novel application of a two-phase extraction of inclusion body preparations with Triton X-114 reduced endotoxin in solubilised E2-T1 to levels suitable for in vivo use without affecting protein yields. Dynamic light scattering analyses showed 37.5% of the protein was monomeric, the remaining comprised of soluble aggregates. Mice immunised with E2-T1 developed a high titre antibody response by ELISA. Western hybridisation analysis showed E2-T1 was recognised by sera from immunised mice and also by several BVDV-E2 polyclonal and monoclonal antibodies.
We have developed a procedure using E. coli to produce soluble E2-T1 protein from IB, and due to their insoluble nature we utilised a novel approach using Triton X-114 to efficiently remove endotoxin. The resultant protein is immunogenic and detectable by BVDV-E2 specific antibodies indicating its usefulness for diagnostic applications and as a subunit vaccine. The optimised E. coli expression system for E2-T1 combined with methodologies for solubilisation, refolding and integrated endotoxin removal presented in this study should prove useful for other vaccine applications.
大肠杆菌中的蛋白质表达可能导致重组蛋白以不溶性包涵体的形式表达。此外，从大肠杆菌中纯化的蛋白质含有内毒素，需要去除内毒素才能用于体内应用。牛病毒性腹泻病毒（BVDV）的结构蛋白 E2 是主要的免疫原决定簇，是亚单位疫苗的理想候选物。E2 蛋白含有 17 个半胱氨酸残基，这给大肠杆菌表达带来了困难。在本报告中，我们概述了一种从包涵体（IB）中成功生产可溶性和无内毒素 BVDV E2 蛋白的方法。
在大肠杆菌中表达 BVDV-E2 蛋白的截断形式（E2-T1）主要导致聚合的不溶性 IB 聚集。使用含有 100mM 二硫苏糖醇的强还原缓冲液可从 IB 聚集体中以高纯度和稳定性溶解 E2-T1。通过透析到含有 0.2%Igepal CA630 的 50mM Tris（pH7.0）中进行复性，得到可溶性但聚集的蛋白质溶液。两相萃取包涵体制剂与 Triton X-114 的新应用可将溶解的 E2-T1 中的内毒素降低至适合体内使用的水平，而不影响蛋白质产量。动态光散射分析表明，37.5%的蛋白质为单体，其余为可溶性聚集体。用 E2-T1 免疫的小鼠通过 ELISA 产生了高滴度的抗体反应。Western 杂交分析表明，E2-T1 被免疫小鼠的血清以及几种 BVDV-E2 多克隆和单克隆抗体识别。
我们已经开发了一种使用大肠杆菌从 IB 生产可溶性 E2-T1 蛋白的方法，由于其不溶性，我们利用 Triton X-114 的新方法有效地去除内毒素。所得蛋白具有免疫原性，可被 BVDV-E2 特异性抗体检测到，表明其在诊断应用和亚单位疫苗方面的有用性。本研究中提出的 E2-T1 的优化大肠杆菌表达系统以及用于溶解、复性和集成内毒素去除的方法，对于其他疫苗应用应该是有用的。
