Enzyme Kinetics - Experiment 12 Isolation Purification and...

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: , , Experiment 12 Isolation Purification and Kinetics of . E coli Alkaline Phosphatase Biochemistry 332L - - 1 28 08 , For this semester we will be doing some projects that carry over from - . week to week as multi part labs The first set of experiments involve the enzyme alkaline phosphatase from Escherichia coli ; we will begin by - . , culturing the K 12 strain of E coli which is a mutant that lacks proper . genetic control of alkaline phosphatase expression This strain overexpresses , the enzyme we are after and should give us a good yield of protein to work . with A strain of bacteria that has this property is said to be constitutive . for whatever protein is overexpressed ( . . . ) Alkaline phosphatase EC 3 1 3 1 is secreted by into the ( . ), periplasmic space pg 236 in your textbook where it cleaves phosphate groups from any proteins in its environment in order that this inorganic . phosphate can be absorbed by the cell for its own use We will be incubating our ( with lysozyme to digest the cell wall you can review - ). this process in section 8 3 of your textbook Recall that - are Gram . negative , Once the cell wall is digested we will subject the cells to a gentle osmotic shock . by adding EDTA Osmotic shock is a change in the osmotic - pressure that should rupture the now weakened outer cell membrane while . ( : leaving the cytoplasmic membrane intact Consider why does adding EDTA ) to our solution result in osmotic shock? Rupturing a cell is called lysis , and this term is also often used to describe the disruption of the outer . membrane even when the inner membrane is left intact , At this point we will have hopefully liberated the material from the periplasmic space without , , . having ruptured the inner cytoplasmic membrane A bacterial cell that has been partially ruptured in this way is called a spheroplast . , , Spheroplasts contain the cytosolic proteins the DNA the , . cellular organelles etc These particles are then sedimented by , centrifugation leaving a supernatant containing only those relatively few . proteins that have been freed from the periplasmic space Considerable . purification from other cell components can be obtained in this way
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Materials At least six hundred mLs of K12 . E coli cell culture - ( . )/ . ( %) 30 mM Tris HCl pH8 0 0 5M 20 sucrose
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Enzyme Kinetics - Experiment 12 Isolation Purification and...

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