Browsing by Subject "Cloning"

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Cloning, expression, and purification of Burkholderia protein targets for diagnostic and vaccine development
(2012-05) McCaul, Kate Christina; Brown, Katherine A.; Kitto, George B.
Burkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. These diseases are endemic mainly in southeastern Asia and northern Australia, but they also pose a bioterrorism threat in the developed world. These diseases have high mortality, partially due to the lack of vaccines and rapid, accurate diagnostic assays. The work discussed here represents a part of a larger project to develop a dependable diagnostic assay for use in both developing endemic areas and the developed world, as well as a subunit vaccine to protect against disease. In this study, several proteins from B. pseudomallei, B. mallei, and the closely related but less virulent B. thailandensis have been cloned, expressed and purified in order to develop highly sensitive and specific diagnostic reagents for the detection of B. pseudomallei and B. mallei in infected patient samples. Protein targets expressed in this study were also used in subunit vaccine development for melioidosis and glanders.
Isolation and characterization of Pisum sativum apyrases, PsNTP9 and PsNTP9-DM, cloned and expressed in Escherichia coli
(2019-02-06) Wallen, Michael Andrew, Jr; Roux, Stanley J.
Adenosine triphosphate (ATP) is widely known as a fuel source for many biochemical processes, and to a lesser degree also as a signaling molecule in plants and animals. When plants are subjected to biotic or abiotic stress or undergoing exocytosis, they release ATP into the extracellular matrix (ECM). The release of ATP sets off a signal transduction pathway, first rapidly increasing the concentrations of cytosolic calcium, reactive oxygen species, and nitric oxide. How these changes specifically influence physiology is the object of much research in both plants and animals. Some of the changes that are affected influence growth and development, stomatal function, and gravitropism. Apyrases and other phosphatases control the concentration of the released nucleotides by breaking phosphate bonds from nucleoside triphosphates and diphosphates. Research aimed at the discovery of receptors, signaling pathway components, and processes has been successful to some extent. There are now known purinergic receptors in both plants and animal cells. We have cloned a truncated version of Pisum sativum (ps) NTP9. We used a pET-22B vector to add a histidine tag and transformed the vector into the BL21 Escherichia coli with a T7 promoter to enable IPTG induction of the LAC operon and expression of the enzyme. The pET-22B vector was incubated in separate samples with BL21 cells. Cells were propagated, and the expression of recombinant proteins PsNTP9, and separately, a double mutant PsNTP9-DM with a second calmodulin-binding domain, were induced ectopically. Cells were broken open by shaking them and mixing them with lysis buffer. Centrifugation was performed to separate the supernatant containing the released apyrases from the particulate wall fraction. The enzymes were purified by affinity chromatography, then their purity was evaluated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). Western blots were performed to verify presence of the apyrases using a commercial anti-histidine antibody to detect PsNTP9 and PsNTP9-DM. Once suitable amounts of our proteins of interest were harvested, we performed Bradford assays to determine the protein concentration of the samples and carried out an apyrase activity assay to determine the specific activity of the purified enzymes and compare it to that of other known phosphatases.