Site-directed mutagenesis of acetylcholinesterase - a tool for studying structure/function relationship (CROSBI ID 104610)
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Kovarik, Zrinka ; Radić, Zoran ; Taylor, Palmer
engleski
Site-directed mutagenesis of acetylcholinesterase - a tool for studying structure/function relationship
Background and purpose: Seven site-directed mutants of mouse acetylcholinesterase (AChE ; EC 3.1.1.8) were constructed in order to examine the structural bases of mechanism of AChE interaction with inhibitors. Mutants were selected based on location in the AChE active site and on possible role in AChE interactions. The mutations combined substitutions in the choline binding site (Y337A, F338A) with the acyl pocket (F295L, F295A, F297I, F297A) and with the residue N-terminal to the catalytic serine (E202Q). Materials and methods: A cDNA encoding mouse AChE truncated at position 548, which yields a monomeric form of the enzyme, was used in mutagenesis as the wild-type cDNA. Mutant DNA was generated by PCR mediated mutagenesis while multiple mutants were generated by subcloning of DNA fragments containing single mutations into the mammalian expression vector, pCDNA3 or pRCCMV. Human embryonic kidney (HEK) cells were transfected, and stable transfectants were generated by selecting transfected cells with G418. Harvests of the media containing the mutant AChE were subjected to purification by affinity chromatography using procainamide affinity resin and decamethonium as elutant. After dialysis of the purified mutant, levels of purity were determined by SDS-PAGE using the endoglycosidase, PGNase F. Results and conclusions: Generated mutants are catalytically efficient enzymes, and can be used as a tool for studying AChE structure/function relationships in the interaction with inhibitors.
acetylcholinesterase; site-directed mutagenesis; mammalian expression system
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