HIV particle assembly is mediated by the Gag protein. Over several years the author has been involved in the characterization of Gag using analytical ultrancentrifuge methodologies. This work was a collaboration developed by Alan Rein and his associate Sid Datta from NCI, in conjunction with external collaborators Susan Krueger and Joseph E. Curtiss. It came to fruition in the publication of two papers in the Journal of Molecular Biology. These papers are listed in the Bibliography and full details of the results can be obtained from these papers. ? ? The Malaria Vacccine Development Branch (MVDB) of NIAID is exploring the use of recombinant Plasmodium falciparum surface proteins for their potential to vaccinate against malaria. Merozoite surface protein 3 (MSP3) is a putative antigen for immunization against malaria, and this has been demonstrated using a recombinant yeast-derived Plasmodium falciparum MSP3 that protected Aotus nancymai monkeys against a virulent challenge infection. In an effort to produce a recombinant MSP3 protein in a scaleable manner, David Narum's laboratory expressed and purified near full-length MSP3 in Escherichia coli (EcMSP3). Purified EcMSP3 formed highly asymmetrically shaped dimers as determined by analytical size exclusion HPLC with in-line multi-angle light scattering and quasi-elastic light scattering detection (QELS) and velocity sedimentation (Rh 7.6 +/- 0.2 nm and 7.3 nm, respectively). Sedimentation equilibrium confirmed the molar mass determined by light scattering. The dimer formation occurred through the leucine zipper-like domain located at the carboxyl-terminus. The surface conformation of MSP3 has been imaged by atomic force microscopy (Albert Jin, LBPS, NIBIB) and the dimer form found to have a star-like molecular structure. This star-like structure would be expected to have considerable hydrodynamic friction, which is consistent with the asymmetrical structure found from sedimentation velocity and QELS. Molecular homology modeling (Darrel Hurt) is in progress for MSP3 as well. This combined biophysical, imaging and molecular homology modeling allows for the development of a conformational model for MSP3. A manuscript is currently in preparation.? ? A number of malaria proteins are being cross-linked and tested as immunogens. To characterize the size distribution of these constructs David Narum's laboratory performs in-line multi-angle light scattering, and we perform sedimentation velocity measurements.
| Shimp Jr, Richard L; Martin, Laura B; Zhang, Yanling et al. (2006) Production and characterization of clinical grade Escherichia coli derived Plasmodium falciparum 42 kDa merozoite surface protein 1 (MSP1(42)) in the absence of an affinity tag. Protein Expr Purif 50:58-67 |
