Native display of complete foreign protein domains on the surface of hepatitis B virus capsids

Abstract

The nucleocapsid of hepatitis B virus (HBV), or HBcAg, is a highly symmetric structure formed by multiple dimers of a single core protein that contains potent T helper epitopes in its 183-aa sequence. Both factors make HBcAg an unusually strong immunogen and an attractive candidate as a carrier for foreign epitopes. The immunodominant c͞e1 epitope on the capsid has been suggested as a superior location to convey high immunogenicity to a heterologous sequence. Because of its central position, however, any c͞e1 insert disrupts the core protein's primary sequence; hence, only peptides, or rather small protein fragments seemed to be compatible with particle formation. According to recent structural data, the epitope is located at the tips of prominent surface spikes formed by the very stable dimer interfaces. We therefore reasoned that much larger inserts might be tolerated, provided the individual parts of a corresponding fusion protein could fold independently. Using the green f luorescent protein (GFP) as a model insert, we show that the chimeric protein efficiently forms f luorescent particles; hence, all of its structurally important parts must be properly folded. We also demonstrate that the GFP domains are surface-exposed and that the chimeric particles elicit a potent humoral response against native GFP. Hence, proteins of at least up to 238 aa can be natively displayed on the surface of HBV core particles. Such chimeras may not only be useful as vaccines but may also open the way for high resolution structural analyses of nonassembling proteins by electron microscopy.

A fusion product of the complete Borrelia burgdorferi outer surface protein A (OspA) and the hepatitis B virus capsid protein is highly immunogenic and induces protective immunity similar to that seen with an effective lipidated OspA vaccine formula

“…However, since the particle-forming ability of such fusion proteins frequently decreased with increasing insert size, the c/e1 loop was thought to have a limited insertion capacity. This assumption was disproved with our finding that CLP are able to display the entire 238-aa green fluorescent protein (GFP) in its native form on their surface [34].…”

Three-dimensional Reconstruction of the Recombinant Type 2 Ryanodine Receptor and Localization of Its Divergent Region 1

“…The cloning and construction of the cDNA encoding the mouse cardiac muscle RyR2 wt have been described previously (20). The cDNA of RyR2 D4365-GFP was designed such that the entire GFP sequence, with Gly-rich spacers on each side (19), is inserted after the Asp-4365 in the DR1.…”

“…To further minimize potential disruptive effects of the insertion on folding of both GFP and RyR2, two Gly-rich spacers were added to flank either side of the GFP (19).…”

“…1 The abbreviations used are: RyR2, type 2 RyR or cardiac RyR; RyR, ryanodine receptor; RyR1 and -3, RyR isoform 1 and 3, respectively; DR1, -2, and -3, divergent region 1, 2, and 3, respectively; EM, electron microscopy; FKBP12.6, 12.6-kDa FK506-binding protein; GFP, green fluorescent protein; GST, glutathione S-transferase; PIPES, 1,4-pipera-reporter protein for monitoring gene expression and protein targeting (16), and it appears to be a good choice as an insertional fusion protein, because its characteristic green fluorescence depends on the proper folding of the protein (17,18). Indeed, this approach has been shown to be feasible for structural analysis of surface-exposed regions of icosahedral viruses by cryo-EM (19). Proteins of the size of GFP (molecular mass 28 kDa) are easily visualized and mapped by image processing, even at moderate (ϳ3-nm) resolution.…”

Fibronectin-binding nanoparticles for intracellular targeting addressed by B. burgdorferi BBK32 protein fragments

“…Earlier it was shown that failure of some chimeric proteins to assemble into VLPs could be explained by the length of the insert; 120 amino acids are usually accepted as a maximum. On the other hand, Kratz et al 36 reported successful insertion of the GFP protein (238 amino acids) in the HBc; thus the structural importance of proper and independent folding of foreign sequences was clearly demonstrated. The potential role of different characteristics in the efficient assembly of VLPs was addressed in numerous studies, such as β-sheet forming properties, the distance between the N and the C-terminus, and volume and hydrophobicity of the amino acids of the insert.…”