Date of Award
Master of Science
The oceans play significant roles in maintaining the climate on Earth and providing nutrients to the whole biosphere. Oceanic microbes produce approximately half of the Earth's oxygen. It has been shown that the populations of microbial communities in oceans are largely regulated by viral infection and thus affect the marine ecosystem. Cafeteria roenbergensis Virus (CroV) is a giant marine virus with a linear, double stranded, and AT-rich DNA genome. The genome of CroV has a size of 744kb and encodes for 544 predicted genes. CroV derived its name from its host, a unicellular marine zooplankton Cafeteria roenbergensis (Cro) that is the major microbial grazer in the ocean. By controlling Cro's population, CroV has a great impact on the marine ecological system. In addition, CroV is a member of the nucleo-cytoplasmic large DNA virus (NCLDV) clade. The presence of genes in NCLDVs encoding enzymes normally only required by cellular organisms for DNA replication, DNA repair, transcription, and translation have stimulated debate over the role played by giant viruses in the evolution of cells. However, despite their ecological and evolutionary importance, limited studies have been reported on the structure of giant marine viruses and their life cycles.
CroV is covered by a protein shell (capsid) comprised of major capsid proteins (MCP) and minor capsid proteins (mCP). The capsid not only protects the genetic material of the virus, but also helps in host-virus recognition, which is the critical initial step for viral infection. This research focuses on obtaining large amount homogenous CroV MCP for future structural studies that will facilitate the understanding of the roles of MCP in the viral life cycle. CroV MCP gene was codon optimized, synthesized, and amplified by PCR followed by cloning into a bacterial expression system in order to obtain purified protein of high homogeneity. Initial trials in the expression vector without protein chaperone failed to obtain soluble MCP. By fusing trigger factor (TF), a bacterial cold shock chaperone, to the N-terminal of the CroV MCP, we obtained soluble TF-MCP. TF facilitates the proper folding of proteins at low temperature in bacteria. After large-scale expression in bacteria, ultra-high purity TF-MCP fusion protein was obtained by affinity chromatography and size exclusion chromatography. Mass spectrum (MS) and dynamic light scattering (DLS) were performed to analyze the molecular weight and hydrodynamic diameter of the fusion protein. Homologous sequence comparison and structural modeling indicate CroV MCP should have a trimeric double-jelly-roll structure as seen in PBCV-1, another giant NCLDV. The MS and DLS results suggested that pure TF-MCP fusion protein is in trimeric form, which indicates the proper folding of the MCP. This has built a strong foundation for the next step; to crystallize MCP and determining its atomic structure. The crystal structure of MCP will provide insights about the virus-host interaction as well as viral infection strategy and genome delivery mechanism.
Received from ProQuest
Chakraborty, Sayan, "Expression And Characterization Of The Major Capsid Protein (MCP) Of A Giant Marine Virus: Cafeteria Roenbergensis Virus" (2013). Open Access Theses & Dissertations. 1598.