| dc.description.abstract | 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. | en |
