Date of Graduation
Doctor of Philosophy in Chemistry (PhD)
Chemistry & Biochemistry
Thallapuranam Krishnaswamy Suresh Kumar
Second Committee Member
Third Committee Member
Fourth Committee Member
With the latest innovations in biological sciences, large quantities of biologically active polypeptides as well as high throughput screening methods to quickly evaluate if these biomolecules potentially have therapeutic, diagnostic, or industrial purposes are required. The synthesis and purification of peptides and small proteins continue to be demanding as the production of high yields through chemical synthesis can involve large costs. On the other hand, there are only few examples of acquiring those biomolecules through cloning and expression in bacterial systems in form of recombinant fusion proteins. Glutathione S-Transferase (GST) is not only a very commonly used affinity tag to increase expression yields, but is also known to enhance the solubility of the protein of interest making it a valuable tool in the pursuit of purifying recombinant proteins. Moreover, multidimensional NMR spectroscopy is a widespread technique to reveal the 3D solution structure of proteins. Yet, obtaining structural information of peptides and small proteins can be difficult. In this context, we have developed a rapid purification of peptides and small proteins by fusing them to GST. The method developed is advantageous over the other reported methods due to its easy one-step purification yielding large amounts of fusion protein. Subsequently, the fusion protein is cleaved enzymatically under mild conditions, and the cleavage products are separated using an efficient heat treatment process. Our results show, the peptide and small protein conformations are not disturbed by the heat treatment. Therefore, our method can be a valuable alternative for the production of various clinically significant small proteins and peptides. Furthermore, we have optimized a method, which allows collecting structural information on protein/ peptide(s) of interest by employing the GST-tagged target protein during the acquisition of NMR data. Our results demonstrate that the affinity tag GST does not affect the quality of NMR data of its fused partner but that the loss of signals in the 1H-15N HSQC spectrum corresponding to the affinity tag is due to the decrease in the T2 relaxation rate upon dimerization as well as the flexibility within the fusion protein caused by the linker located between GST and the target protein.
Kachel, Wibke Beatrice, "Applications of the GST- Affinity Tag in the Purification and Characterization of Proteins" (2016). Theses and Dissertations. 1625.