Recombinant expression of the cowpea chlorotic mottle virus

Sammanfattning: All viruses are composed of both genetic material and a capsid. The capsid is composed by identical protein subunits and the purpose is protection of the genetic material, in this case RNA. This project is about cowpea chlorotic mottle virus, also called CCMV, which is a plant virus and the disassembly and reassembly mechanisms of the capsid are investigated to understand how the capsid proteins associate to create capsids. These mechanisms are suitable to use as a model system to study protein self-assembly. The capsid is able to disassembly and removes the genetic material. The capsid proteins can then assembly to an empty capsid without any genetic material. The goal with this project is to develop a method to recombinant express CCMV and isolate dimers that are appropriate for assembly. Previous research has tried to take advantages of a binding his-tag to facilitate the purification. These experiments have been unsuccessful. For that reason, this project doesn´t use a his-tag. Plasmids containing the gene of interest were recombinant expressed in E.coli and purified with ammonium sulphate precipitation followed by gel filtration. Dynamic light scattering and mass spectrometry were performed on fractions from the gel filtration. The solution conditions were changed to control assembly of capsid proteins. Dimers were isolated and experiments with a 50/50 mix of dimers and assembly buffer with different pH and incubation times were performed to create capsids. A 50/50 mix of dimers and assembly buffer with pH 4.5 and incubation for 30 minutes probably created capsids. After the expression was the protein found in the soluble part of the clarified lysate. Previous research has found the protein in the inclusion bodies. Further experiments should try to optimize the purification and also the solution conditions. The assembly-competent CCMV dimers can be recombinant expressed and isolated and thus useful in further biophysical experiments.