The Nup62 Coiled-Coil Motif Provides Plasticity for Triple-Helix Bundle Formation.

The central transport channel of the vertebrate nuclear pore complex (NPC) consists of nucleoporins: Nup62, Nup54, and Nup58. The coiled-coil domains in α-helical regions of these nucleoporins are thought to be crucial for several protein-protein interactions in the NPC subcomplexes. In this study, we determined the crystal structure of the coiled-coil domain of rat Nup62 fragment (residues 362-425) to 2.4 Å resolution. The crystal structure shows the conserved coiled-coil domain as a parallel three-helix bundle for the Nup62(362-425) fragment. On the basis of our size exclusion chromatography coupled to multiangle light scattering analysis and glutaraldehyde cross-linking experiments, we conclude that the Nup62(362-425) fragment displays dynamic behavior in solution and can also exist in either homodimeric or homotrimeric states. Our comparative analysis of the rat Nup62(362-425) homotrimeric structure with previously reported heterotrimeric structures [rat Nup62(362-425)·Nup54(346-407) and Xenopus Nup62(358-485)·Nup54(315-450)·Nup58(283-406) complexes] demonstrates the structural basis for parallel triple-helix bundle formation for Nup62 with different partners. Moreover, we show that the coiled-coil domain of Nup62 is sufficient for interaction with the coiled-coil domain of rat Exo70, a protein in an exocyst complex. On the basis of these observations, we suggest the plausible chain replacement mechanism that yields to diverse protein assemblies with Nup62. In summary, the coiled-coil motif present in Nup62 imparts the ability to form a homotrimer and heterotrimers either with Nup54 or with Nup54-Nup58 within the NPCs as well as with Exo70 beyond the NPCs. These complexes of Nup62 suggest the crucial role of the coiled-coil motifs in providing plasticity to various modular assemblies.

Expression of rabies glycoprotein and ricin toxin B chain (RGP-RTB) fusion protein in tomato hairy roots: a step towards oral vaccination for rabies.

Transgenic hairy roots of Solanum lycopersicum were engineered to express a recombinant protein containing a fusion of rabies glycoprotein and ricin toxin B chain (rgp-rtxB) antigen under the control of constitutive CaMV35S promoter. Asialofetuin-mediated direct ELISA of transgenic hairy root extracts was performed using polyclonal anti-rabies antibodies (Ab1) and epitope-specific peptidal anti-RGP (Ab2) antibodies which confirmed the expression of functionally viable RGP-RTB fusion protein. Direct ELISA based on asialofetuin-binding activity was used to screen crude protein extracts from five transgenic hairy root lines. Expressions of RGP-RTB fusion protein in different tomato hairy root lines varied between 1.4 and 8 µg in per gram of tissue. Immunoblotting assay of RGP-RTB fusion protein from these lines showed a protein band on monomeric size of ~84 kDa after denaturation. Tomato hairy root line H03 showed highest level of RGP-RTB protein expression (1.14 %) and was used further in bench-top bioreactor for the optimization of scale-up process to produce large quantity of recombinant protein. Partially purified RGP-RTB fusion protein was able to induce the immune response in BALB/c mice after intra-mucosal immunization. In the present investigation, we have not only successfully scaled up the hairy root culture but also established the utility of this system to produce vaccine antigen which subsequently will reduce the total production cost for implementing rabies vaccination programs in developing nations. This study in a way aims to provide consolidated base for low-cost preparation of improved oral vaccine against rabies.