CaYPD1 purification(1)

CaYPD1 purification(1) - CaYPD1 purification-1 3 Aim and...

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CaYPD1 purification-1 - 3 Aim and Importance: The aim of these experiments was to prepare the solution of protein CaYPD1 for purification prior to SDS PAGE. First the column was packed with 1- 1.5 ml Ni-NTA agarose beads. Second to obtain the protein CaYPD1, the BL21(DE)3 cell that has expressed the CaYPD1 protein was broken down using sonication. Sonication exploits ultrasonic energy to disrupt both the cell wall and cell membrane to release the contents into the solution. Followed by a combination of biochemical and mechanical techniques such as including various types of filtration and centrifugation were used to separate different cell compartments and organelles. The Ni-NTA column was washed with buffers 1-5 and the flow-through collected in eppendorf tubes. The importance of these experiments is hidden in the various steps that students need to have detail knowledge of to understand the process being followed. Background: Candida albicans, one of the top four causes of nosocomial infectious diseases in humans, is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in humans. Candida albicans contains the protein CaYPD1, which plays an important role in the virulence and cell adaptation of Candida albicans by functioning in the histidine-aspartate signal transduction system, the process by which an extracellular signaling molecule activates a membrane receptor creating a response, reacts to oxidative stresses. CaYPD1 gene has been cloned into pET16b plasmid and pET21a vectors by Dr. Alla Kasara in Dr. Ann West’s Laboratory form the University of Oklahoma. The words vector represents Plasmids used in genetic engineering. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to make many copies of or express particular genes . The pET-21a-d(+) vectors carry an N-terminal T7•Tag sequence plus an optional C-terminal His•Tag sequence. The pET-16b vector includes an N-terminal His•Tag sequence, a Factor Xa site and three cloning sites. These vectors
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CaYPD1 purification(1) - CaYPD1 purification-1 3 Aim and...

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