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Characterization of Truncated Tumor-Associated NADH Oxidase (ttNOX)Bacterial, plant and animal cells possess novel surface proteins that exhibit both NADH oxidation (NOX) or hydroquinone and protein disulfide-thiol interchange. These enzymatic activities alternate to yield oscillating patterns wjth period lengths of approximately 24 minutes. The catalytic period of NOX proteins are temperature compensated and gravity responsive. We report the cloning, expression and characterization of truncated tumor-associated NADH oxidase (ttNOX), in which the membrane spanning region has been deleted. The cDNA (originated from HeLa cells) was cloned into pET-34b and pET-14b (Novagen) vectors for E. coli expression. Optimized expression and purification protocols yielded greater than 300mg per liter of culture with greater than 95% purity. Circular dichroism data was collected from a 2.7mg/ml solution in a 0.1mm cuvette with variable scanning using an Olis RSM CD spectrophotometer. The ellipticity values were scanned from 190 to 260nm. The spectra recorded have characteristics for alpha proteins with band maxima at 216nm and a possible shoulder at 212nm at 12OC and 250 C. Protein crystal screens are in progress and, to date, only small crystals have been observed. The regular periodic oscillatory change in the ttNOX protein is indicative of a possible time-keeping functional role. A single protein possessing alternating catalytic activities, with a potential biological clock function, is unprecedented and structural determination is paramount to understanding this role.
Document ID
20030001591
Document Type
Conference Paper
Authors
Karr, Laurel J. (NASA Marshall Space Flight Center Huntsville, AL United States)
Malone, Christine C. (Universities Space Research Association Huntsville, AL United States)
Burk, Melissa (Morgan Research Corp. Huntsville, AL United States)
Moore, Blake P. (Alabama Univ. Huntsville, AL United States)
Achari, Aniruddha (Raytheon Co. Huntsville, AL United States)
Curreri, Peter A.
Date Acquired
August 21, 2013
Publication Date
January 1, 2002
Subject Category
Life Sciences (General)
Meeting Information
XIX Congress of the International Union of Crystallography(Geneva)
Distribution Limits
Public
Copyright
Work of the US Gov. Public Use Permitted.