The University of Georgia College of Veterinary Medicine

Georgia Veterinary Scholar Program

Georgia Veterinary Scholar	Faculty Mentor

Jeff Stortz University of Georgia Class of 2004	Dr. Zhen Fu

Purification of rabies virus nucleoprotein using bacteria and baculovirus systems

The rabies virus consists of a nonsegmented, single stranded, negative sense RNA genome that codes for five proteins: nucleoprotein (N), phosphoprotein (P), matrix (M), glycoprotein (G), and RNA polymerase (L). One function of the N protein is to encapsidate leader RNA produced during transcription, and this is thought to play a role in switching the virus from transcription mode to replication. To study the structure and function of the N, we expressed the N in a baculovirus system as well as a bacteria system. In the baculovirus system, insect cells were infected with baculovirus expressing the N protein. An anti-N antibody affinity column was utilized to purify the protein. In the bacteria system, bacteria containing plasmids with the N gene were cultured and harvested. A Talon metal affinity column was used to purify the N protein through the binding of a His tag inserted in the plasmid with a cobalt resin. Analysis of the purified N protein by SDS- PAGE revealed impurities in the N preparation purified using either of these methods. Thus, we employed gel filtration column to further separate the N protein from the impurities. This column separates substances by eluting them in different fractions depending on their molecular weight. This method greatly reduced the impurities present as verified by SDS- PAGE. These purified N proteins will be used for crystallography and will help us better understand its structure and function in relation to the processes of rabies transcription and replication.