The production of normal and variant human glucose-6-phosphate dehydrogenase in cos cells |
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Authors: | P J Mason T J Vulliamy N S Foulkes M Town B Haidar L Luzzatto |
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Institution: | Department of Haematology, Royal Postgraduate Medical School, London, England. |
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Abstract: | Full-length cDNA coding for human glucose-6-phosphate dehydrogenase (G6PD) was inserted into a eukaryotic expression vector containing the immediate early promoter of cytomegalovirus. When this plasmid was introduced into cos cells by transfection it led to the production of high levels of human G6PD. cDNAs containing mutations found in G6PD-deficient individuals were constructed by in vitro mutagenesis and expressed in the same system. Characterization of the G6PD proteins obtained in this way confirmed the primary structure inferred for the variant enzymes. An enzyme in which lysine-205 had been mutated to threonine was produced and found to have no G6PD activity, proving that this lysine residue is essential for enzyme activity in human G6PD. |
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