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Date of Award
2010
Degree Type
Thesis
Degree Name
Bachelor of Science
Department
Biology
First Advisor
Dr.Kenneth Murray
Abstract
Dr. Kenneth Murray, Ph.D. Assistant Professor of Biology Ribonuclease P (RNase P) is an essential and ubiquitous ribonucleoprotein enzyme primarily responsible for cleaving 5' leader sequences during tRNA maturation. RNase P comprises one essential RNA, and one protein subunit in eubacteria, five proteins in archaea, and ten in humans. Due to its homology to human RNase P, its higher stability, and simpler structure; extensive studies have been conducted utilizing the enzyme from the archaeal hyperthermophile, Pyrococcus furious (Pfu). Previous studies identified only four protein subunits associated with the archaeal RNase P. This fourprotein reconstituted particle, however, had an optimal temperature of 55°C, compared to the optimal 70°C of the wild type RNase P. Additional probing of the organism's genome database revealed a fifth RNase P protein subunit, RPP38. To facilitate further investigations of Pfu RNase complexes, we sought to develop a protocol for the purification ofRPP38. Our results, presented herein, represent the first known expression.purification protocol developed for RPP38. Briefly, we synthesized an N-terminal6x-His RPP38 fusion construct, reengineered to contain a Tobacco Etch Virus (TEV) protease cleavage site. Purification was achieved via immobilized metal affinity chromatography and reversed phase high performance liquid chromatography. Following purification the 6X-His affinity tag was removed via TEV cleavage, thus regenerating the native RPP38 protein. Purity and identity of RPP38 were confirmed by sodium dodecylsulfate - polyacrylamide gel electrophoresis and mass spectrometry, respectively. Our work is expected to contribute to our understanding ofRNase P function and tRNA maturation by providing an efficient, facile technique to express and purify Pfu RNase protein RPP38 as a means to facilitate structural and functional analyses.
Recommended Citation
Lee, Kimberley, "Novel Purification of Archaeal Rnase P Protein, Pfu Rpp38, for Nmr Studies" (2010). Department of Biological Sciences - Undergraduate Honors Theses. 26.
https://digitalcommons.fiu.edu/bio_honors/26
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