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Cloning and site-directed mutagenesis of alkaline phosphatase from E. coli

Alkaline phosphatases (AP) are nonspecific phosphomonoester hydrolases. They are found in most species from bacteria to man indicating an involvement in fundamental biological processes. All APs are mulitmeric metalloproteins found outside the cell, in mammals attached to the membrane via a phosphatidyl inositol anchor or residing in the periplasmic space of bacteria. Kinetic and structural properties of AP have been thoroughly investigated using various spectroscopic and kinetic techniques. AP displays a characteristic kinetic behaviour, commonly described as the negative cooperativity or the half-of-the-sites reactivity. Most unresolved questions are related to conformational changes in the catalytic cycle, allosteric interactions and their influence on the catalytic efficiency. AP from E. coli is often used as a model enzyme system to study the role of metal ions in catalysis, and for the investigations of the characteristic properties of oligomeric enzymes such as negative cooperativity or the half-of-the-sites reactivity, and interactions between subunits. In addition, it could be used as a model to investigate the potential advantage of dimeric enzyme, with such kinetic properties, over monomeric enzymes. Our study is focused on understanding the role and nature of subunit interactions in the catalytic mechanism of AP from E. coli. To investigate the role of specific amino acid in establishing communication between the monomers, site-directed mutagenesis on the E. coli alkaline phosphatase gene (phoA) has been conducted. PhoA was amplified from genomic DNA using the polymerase chain reaction, and inserted into a pET expression vector. Overproduction of wild-type PhoA was confirmed by SDS-PAGE and the activity assay. The mutant enzyme was created by replacement of Thr81, located at the subunit interface and hydrogen-bonded to its counterpart of the other subunit, with alanine.

alkaline phosphatase; site-directed mutagenesis

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