Doctor of Philosophy (PhD)



Document Type



When specific chlamydia chaperone 4 (Scc4) and specific chlamydia chaperone1 (Scc1) are co-expressed in E. coli, they form a soluble and stable Scc4:Scc1 complex. In an attempt to selectively label one protein in the complex in vivo with isotopes for nuclear magnetic resonance (NMR) studies, it was observed that sequential expression of Scc4 and Scc1 in E. coli resulted in no complex or an unstable complex depending on the culture medium used. These results, taken together with previous experiments to form the Scc4:Scc1 complex in vitro, suggest that Scc4 and Scc1 assemble co-translationally. In Chlamydia trachomatis, the co-translational assembly would require the simultaneous expression of Scc4 and Scc1 in the mid-to-late stages to form the chaperone complex, Scc4:Scc1, suggesting that Scc4 in the early-to-mid stages does not switch from its RNAP-binding function. A method was developed to express the mutant protein of Scc4, Scc4(V95A), with a cleavable purification tag that minimizes extra amino acids. The proteins were expressed with N-terminal His-tags, human rhinovirus 3C protease recognition sequence, and NusA tags for purification and solubility for the wild-type and mutant sequences. After purification using Ni-IMAC, the fusion proteins were treated with HRV3C protease. This resulted in a mixture of the cleaved tags and the proteins with their sequence modified by only two extra amino acids at their N-termini, Gly-Pro. Ni-IMAC and cation exchange chromatography were assessed to purify the Scc4 proteins from the cleaved, His-NusA-tag, and HRV3C protease with moderate results of 25% recovery. The feasibility of using the thermal shift assay (TSA) to study protein-ligand binding with the Scc4:Scc1 complex and the Scc4 protein was evaluated by setting up TSA to find appropriate conditions. Our observations demonstrated that TSA is not viable for Scc4 since we did not find conditions where Scc4 has an ideal melting curve. However, for the Scc4:Scc1 complex, TSA can be considered a feasible screening method due to the generated ideal melting curves under studied conditions. Future work would be to use TSA with the complex to screen small (drug-like) molecules.



Committee Chair

Garno, Jayne C.

Available for download on Saturday, April 03, 2027