Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates

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.

Development of nanoparticle based vaccine delivery systems

“…[40][41][42] In our laboratory, generation of soluble and endotoxin-free oE2 using an E. coli expression system has been well established. [43,44] The adsorption capacity of proteins to the nanoparticles is greatly dependant on the physicochemical characteristics of the proteins as well as the nanoparticles. Proteins with the hydrodynamic diameter that is smaller than the pore diameter can easily enter the mesopores and show higher loading capacity, while the ones with larger diameter adsorb on the outer surface of the nanoparticles.…”

“…They can be structurally modified to be in different shapes such as rods and have chirality. [40] Since the discovery of the M41S family of mesoporous materials in 1992, a number of different mesoporous silica nanoparticles have been invented, which include MCM-n [41], SBA-n (Santa Barbara Amorphous) [42], IBN-n (Institute for Bioengineering and Nanotechnology) [43] and FDU-n (Fudan University). [44] Different organic functional groups such as amino (-NH 2 ), thiol (-SH), vinyl (-CH=CH 2 ) and phenyl (-C 6 H 5 ) can be incorporated into or onto the walls of the silica particles.…”

“…[30] In addition many research studies have focussed on synthesis, biocompatibility and biodistribution of MSNs. [15,[31][32][33][34][35][36][37][38][39][40][41][42][43][44][45] Various studies have shown that MSNs are excellent vehicles for gene and drug delivery because of their ease of synthesis, surface functionalisation, excellent in vivo biocompatibility as well as good thermal and chemical stability. [40,46,47] Additionally, MSNs are capable of controlling the release of drugs or proteins depending on their size, shape and surface modification.…”

“…[43] The use of nanoparticles as delivery systems can improve protein and peptide stability, enhance protection and facilitate presentation of antigen to the immune system. [40,44] Protein/antigens on adsorption onto the nanoparticles, can either adsorb on the surface or internal pores of the nanoparticles; site at which the proteins bind to the nanoparticles greatly depend on the type and properties of the target proteins and nanoparticles (Fig 3.1).…”

“…[68] HMSNs have been synthesised using a sol-gel/emulsion method with modifications to adsorb antigens; [69] in addition, HMSCs with novel capsular morphology have been synthesised using the cetyltrimethyl ammonium bromide (CTAB) micellar assembly in cholesterol emulsion. [43] Synthesising materials with large pore (~30 nm to 50 nm), small particle size (50 nm to 200 nm) and tunable surface properties will allow sufficient space for proteins and peptides to adsorb and release the payload at the desired site, and hence remains the area of intense focus.…”