Isolation of Alkaline Phosphatase from E. coli
Josh Brooks
Department of Chemistry and Biochemistry, McMurry University, Abilene TX 79697
Abstract:
In this experiment our goal was to analyze alkaline phosphatase from E. coli .In order to do this the purification of a protein from E. coli many steps were done through multiple weeks of assays. Finally, once the desired protein was pure a Bradford and a gel can be run to be sure that the protein was indeed purified.
Introduction:
In order to purify the protein many step are needed and the analyte should be evaluated by measuring the concentration and the activity of the protein. Physical and biological properties are used to help determine purity. These factors can be used to distinguish the analyte from other proteins. Located in the periplasmic space, the space between the cytoplasmic member and the outer membrane, alkaline phosphatase is cleaved and provides the cell with inorganic phosphate. To get a large enough sample of alkaline phosphatase the K12 mutant of E. coli is used. This mutant is unregulated in the cell because it lacks a way to control alkaline phosphatase production. Alkaline phosphatase is best used in acidic conditions and is very stable at high temperatures. By taking advantage of these two properties alkaline phosphatase can be isolated from the other proteins in E. coli. The overall goal is to reduce the total amount of protein but increase the specific activity of the desired protein. The experiment will also determine which particular method, assay, worked best.
Methods and Materials