A novel bipartite SNARE protein affinity model
Foy, Gail Julia
MetadataShow full item record
Protein purification is a fundamental step in obtaining pure proteins for research, therapeutic, agricultural or industrial applications. To facilitate affinity chromatography, solubility and detection, recombinant proteins are often expressed as fusion proteins, where an additional ‘tag’ sequence is incorporated to provide the target protein with unique biochemical characteristics. For example, the commonly used poly-histidine or glutathione S-transferase affinity tags allow the fusion protein to be purified using the tag’s high affinity for an immobilised ligand. Unfortunately, these affinity purification procedures commonly require further downstream purification exploiting differences in protein charge and size to obtain pure proteins. This extensive, multi-step processing of recombinant protein can considerably lower yield and increase production costs. Soluble NSF-attachment protein receptors (SNARE) proteins are investigated here as potential affinity tag and ligand components, in an attempt to design a fusion tag which could function as a one-step high-yield purification process. SNARE proteins display very high affinity and specificity for each other, interacting during exocytosis to create a coiled-coil complex, with unique thermal and chemical resistance. However, their tripartite nature does not make them suitable candidates in their native forms. In this project, I used the neuronal SNAREs to generate a two-component system, which involved the design of a chimeric protein. The design was tested and alternate tag-ligand constructs evaluated to find the optimal configuration of tag and ligand. The results demonstrate that a bipartite purification system composed of cytosolic synaptobrevin, and a chimeric ligand containing SNAP 25 and syntaxin 1A motifs successfully purified tagged enhanced green fluorescent protein (EGFP) from cellular extract. Binding of the tag to the ligand also resulted in a novel binding characteristic in anionic detergent, which could be exploited to purify denatured proteins. In addition, a synaptobrevin tagged human rhinovirus 3C protease was used to successfully cleave GST tagged synaptobrevin, which facilitated the acquisition of untagged target protein. This purification model has the potential to offer a high yield, elevated purity of target protein, and it is anticipated that the research reported in this thesis be further explored in order to quantify the affinity of the tag variants developed, identify optimum expression and purification conditions of the system, and investigate means by which a purification column could be regenerated.