The suspension was ultrasonicated five times at 22?kHz at 15-s intervals on ice. of preS1(20C47) and preS1phil sequences demonstrated remarkable immunogenicity. The same epitope added to the original C-terminus of the HBc protein did not induce a notable AT7519 HCl level of anti-preS1 antibodies. HBcG vectors may contribute to the further development of versatile HBc VLP-based vaccine and AT7519 HCl gene therapy applications. [13, 14] and [15, 16]. The HBc protein consists of two linearly separated domains: (i) the N-terminal self-assembly (SA) domain at amino acid (aa) residues 1C140, which is necessary and sufficient for the protein to self-assemble and result in the structure revealed by X-ray [11], and (ii) the protamine-like arginine-rich C-terminal (CT) domain at aa 150C183 [17], AT7519 HCl whose three-dimensional structure is unresolved. The SA and CT domains are separated by a hinge peptide 141C149 [18, 19]. The SA domain involves the so-called major immunodominant region (MIR), the most protruding aa residues 78C82 of which are located on the tips of the HBc spikes [11]. The MIR AT7519 HCl is generally used for the insertion of foreign B cell epitopes to maximally expose these epitopes on the VLP surface and consequently provide the most efficient immunogenic activity (for review see [4C6]). During HBV life cycle, the CT domain is primarily responsible for the encapsidation of the 3.5-kilobase pregenomic HBV mRNA, which is converted further into partially double-stranded AT7519 HCl HBV DNA (for a recent review see [20]) and is dispensable for self-assembly [21]. Therefore, so-called HBc? particles fully deprived of the CT domain or carrying shortened CT domain fragments are highly efficiently synthesised in bacteria and are consequently often used as the preferred HBc carriers [22]. The nucleic acid-binding sites in the CT domain are organised into four arginine blocks [23] that are buried within HBc VLPs [24]. Although some data demonstrate that CT domain elements may appear on the HBc VLP surface [25C27], the C-terminal insertions of foreign epitopes, in contrast to the MIR and N-terminal insertions, demonstrate generally low immunogenicity in experimental animals (for more detail see [4, 5, 28]). However, the extremely high capacity of the C-terminal insertions [29] has inspired further attempts to elucidate their potential applicability. In this study, we constructed a novel class of HBc VLP carriers, so-called HBcG vectors, in which arginine residues of the CT domain are fully or partially replaced by glycine residues. The elimination of positively charged CT stretches in the HBcG carriers prevents the encapsidation S1PR2 of bacterial RNA by cultivation in and allows the exposure of a C-terminally inserted model epitope, namely, the major epitope of the HBV preS1 sequence, onto the outer surface of HBcG-derived VLPs. This exposure markedly improves the immunogenicity of the inserted epitope in experimental animals. Materials and Methods Bacterial Strains Two strainsK802 (F? rK? mK+for 30?min, the soluble proteins were precipitated with 10?% ammonium sulphate at 4?C for 1?h, followed by centrifugation at 10,000for 30?min. VLPs in the supernatant were precipitated with 35?% ammonium sulphate at 4?C overnight, followed by centrifugation at 10,000for 30?min. The sediment was dissolved in 15?mL of PBS buffer containing 0.5?M urea and 50?M PMSF and subjected to size-exclusion chromatography on a Sepharose 4 Fast Circulation (GE Healthcare, Sweden) 320?mL column (25??850?mm) at a flow rate of 0.5?mL/min. The semi-preparative purification the HBcG-S1phil for the detailed immunological characterisation was performed as follows: 9?g of wet fresh K802 cells was incubated for 30?min on snow in 36?mL of lysis buffer containing 50?mM TrisCHCl, pH 8.0,.