Date of Award
2015
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Biological Sciences
Committee Chair
Joseph D. Ng
Committee Member
Debra M. Moriarity
Committee Member
Roy D. Magnuson
Subject(s)
Nucleases.
Abstract
A putative nuclease from Thermococcus thioreducens was expressed in E. coli BL21 (DE3)-Rosetta cells (6.2 g/l). The recombinant protein (TthNuc) was purified using heat precipitation, anion exchange chromatography, and size exclusion chromatography to a purity of 99%. A BLASTp search of the NR database found TthNuc to be 85% identical to an uncharacterized, RecJ endonuclease from the same genus. TthNuc exhibits in vitro divalent metal ion dependent catabolism of single and double stranded DNA. TthNuc was qualitatively shown to have in vitro nuclease activity between 37°-95°C and above pH 8.4 at 75°C. A three dimensional similarity search of the PDB database found a crystal structure of a nano-rnase (NrnA) from the DHH protein family with 88% query coverage and 22% identical residues with the same domain spatial organization (DHH-DHHA1) as TthNuc. The TthNuc amino acid sequence contains the five essential motifs which characterize the DHH protein family. Also, data suggests TthNuc is a nuclease that is more functionally related to NrnA. TthNuc is a DHH nuclease that should be considered a NrnA-like enzyme until future studies determine its proper classification.
Recommended Citation
Pagan, Jayson Isaza, "Identification of a putative nuclease from Thermococcus Thioreducens" (2015). Theses. 115.
https://louis.uah.edu/uah-theses/115