Abstract
BackgroundProtein 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).ResultsThe 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.ConclusionWe 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.
Highlights
Protein expression in Escherichia coli may result in the recombinant protein being expressed as insoluble inclusion bodies
We describe a novel process for the removal of endotoxins from inclusion bodies (IB) aggregates using Triton X-114 phase-partitioning to maximise E2-T1 yields
We have shown that E2-T1 protein produced in E. coli was recognised by antibodies raised in goat and sheep against whole Bovine Viral Diarrhoea Virus (BVDV) and by various BVDV-specific monoclonal antibodies (Figure 4) indicating the usefulness of E2-T1 for diagnostic applications such as monitoring the serological status of animals which are the natural host of BVDV
Summary
Protein expression in Escherichia coli may result in the recombinant protein being expressed as insoluble inclusion bodies. Mammalian and insect cell line expression of proteins have the advantage of producing proteins with correct conformation and post-translational modifications such as glycosylation, but generally yields are lower than Escherichia coli systems. The use of E. coli based expression systems is hindered by the fact that the recombinant proteins are generally expressed at high yields the resulting proteins are often insoluble and lack post-translational modifications [8]. Expression of glycosylated proteins in E. coli has been previously reported, Chia et al [14] demonstrated the successful generation of neutralising antibodies to the envelope protein (E) of Japanese Encephalitis Virus (JEV), a member of the Flavivirus family. Das et al [15] demonstrated monoclonal antibodies generated against E. coli expressed Ebola virus antigen recognised the glycosylated antigen expressed in mammalian cells
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