Abstract/Details

Applications of the GST- Affinity Tag in the Purification and Characterization of Proteins

Kachel, Wibke Beatrice.  University of Arkansas. ProQuest Dissertations Publishing, 2016. 10137849.

Abstract (summary)

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 STransferase (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.

Indexing (details)


Subject
Cellular biology;
Biochemistry
Classification
0379: Cellular biology
0487: Biochemistry
Identifier / keyword
Pure sciences; Biological sciences; Biomolecules; Glutathione S transferase; Polypeptides
Title
Applications of the GST- Affinity Tag in the Purification and Characterization of Proteins
Author
Kachel, Wibke Beatrice
Number of pages
145
Degree date
2016
School code
0011
Source
DAI-B 78/01(E), Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
978-1-339-93460-0
Advisor
Kumar, Thallapuranam Krishnaswamy Suresh
University/institution
University of Arkansas
Department
Chemistry
University location
United States -- Arkansas
Degree
Ph.D.
Source type
Dissertation or Thesis
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
10137849
ProQuest document ID
1819561221
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
https://www.proquest.com/docview/1819561221/BEB1D89FB36C4165PQ/41