Date of Award

4-2016

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Nikolai R. Skrynnikov

Committee Chair

Nikolai R. Skrynnikov

Committee Member 1

William A. Cramer

Committee Member 2

Chittaranjan Das

Abstract

NMR spectroscopy has recently become a promising field for protein characterization and dynamic studies. As the technology and pulse sequences improve for tracking proteins, a greater demand for developing effective purification protocols to produce NMR grade protein samples have arisen. This thesis explores two proteins: histone H4 tail and Mucin 1; Two very different proteins that require different methods of expression and purification to achieve a high enough yield for NMR analysis. H4 is a water soluble protein that weighs ~2.7 kDa, and has no extinction coefficient. Since the protein is too small for many methods of expression and purification, H4 was attached to thioredoxin and a His tag, purified via Ni affinity column, the tags were cleaved with thrombin, and the resulting H4 was purified via size exclusion. Mucin 1 is a transmembrane protein, and was hard to express within E. coli cells. Adding a thioredoxin tag, and switching to C43(DE3) strain of cells encouraged expression of the protein. The protein was then suspended in 8M Urea, and purified via size exclusion, and thrombin cleavage to remove the thioredoxin. These two different methods of purification provide great insight on how to purify many kinds of proteins for NMR analysis, and is critical knowledge for any protein NMR specialist.

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