Developing Self-assembling Nanocages with Naturally Occurring Bacterial Peptides
Dridge, Charlotte Louise
Thesis or dissertation
University of Exeter
The model selenate respiring bacterium Thauera selenatis produces selenium deposits on a nano-scale during selenate respiration. Selenium deposits have been isolated from Thauera selenatis growth medium, analysed and found to be red solid particles with a diameter of approximately 130 nm. Analysis of the growth medium has suggested that selenium deposits are exported out of the cell, in a process that is facilitated by a key protein termed the selenium export factor, SefA. This protein has been expressed in Escherichia coli and the two recombinant His-tagged forms (His-SefA and His-SefA-His) have been purified. It has been suggested that proteins may influence selenium nanoparticle assembly. Debieux and co-workers investigated the size of selenium nanospheres produced in vitro in the presence of His-SefA and found that they were approximately 300 nm in diameter. This is significantly different to those produced during the growth of Thauera selenatis, and so the effect of His-tagging the SefA protein on the selenium nanoparticles produced was investigated. A non-His-tagged construct of the SefA protein, rSefA, was produced by proteolytic cleavage of the N-terminal His-tag from His-SefA using thrombin. This resulting protein with no His-tags was expected to produce selenium nanospheres most similar to those produced in Thauera selenatis during selenate respiration. Nanoparticles of approximately 130 nm in size, similar to those produced in the bacterium, were produced in the presence of 10 μg/ml rSefA. The size of the nanoparticles was determined using Nanoparticle Tracking Analysis in collaboration with NanoSight Ltd. Selenium nanospheres produced in the presence of His-SefA showed a decrease in particle size as the protein concentration increased. By contrast, those produced in the presence of rSefA showed an increase in particle size as the protein concentration increased. In the presence of His-SefA-His changing the protein concentration had no effect on the size of the selenium particles produced.
MbyRes in Biosciences