YZGD pyridoxal phosphatase from P. thiaminolyticus; subcloning, expression, and purification for x-ray crystallography structure determination

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Title: YZGD pyridoxal phosphatase from P. thiaminolyticus; subcloning, expression, and purification for x-ray crystallography structure determination
Author: Strassner, Amanda M.
Abstract: The HAD superfamily contains enzymes that catalyze carbon or phosphate transfer reactions. One family within the HAD superfamily is the p-nitrophenyl phosphatase (PNPPase) family. Members of this family are all phosphatases that cleave the substrate analog pnitrophenyl phosphate, in addition to their biologically significant substrate. Interestingly, among the small number of family members that have been characterized, there is a wide variety of biologically relevant activities. YZGD from Paenibacillus thiaminolyticus has pyridoxal phosphatase activity, catalyzed by a HAD superfamily domain, as well as a CDP-alcohol/sugar nucleotide hydrolase activity, catalyzed by a Nudix hydrolase domain. Determining the structure of this enzyme would result in valuable information to contribute to our knowledge and understanding of the HAD superfamily and the Nudix hydrolase superfamily. Sufficient quantities of sufficiently pure YZGD are necessary to perform x-ray crystal structure determination. A histidine tag was added to the enzyme so that it can be purified by nickel affinity chromatography. This was done by cloning the gene into pET19b, a plasmid containing a sequence encoding a string of ~10 histidines, and expressing the protein in Escherichia coli BLR(DE3). The tagged YZGD expresses well, is soluble, and retains full activity. It appears to be completely purified by nickel affinity chromatography. This will now allow YZGD to be purified in large quantities, crystallized, and its structure determined.
Record URI: http://hdl.handle.net/1850/6894
Date: 2008-05

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