High-level expression of human asparagine synthetase and production of monoclonal antibodies for enzyme purification.
In order to obtain large quantities of extremely pure human asparagine synthetase for detailed kinetic and structural studies, its gene was cloned into a 2mu plasmid (pBS24.1GAS) suitable for replication in a Saccharomyces cerevisiae cir0 strain (AB116). In this construct, the transcription of the asparagine synthetase gene is regulated by the alcohol dehydrogenase II/glyceraldehyde-3-phosphate dehydrogenase promoter, which is subject to glucose repression. The expression of the enzyme was allowed to take place in yeast minimal medium containing D-galactose as the only sugar nutrient. Eleven monoclonal antibodies to recombinant human asparagine synthetase were produced and one of them was selected to make immunoaffinity resins. After single-step immunoaffinity chromatography, more than 1.2 mg of homogeneous enzyme was obtained from the total cell extract from a 100-ml yeast culture. The yield of pure enzyme was over 100-fold higher than that of a previously reported yeast expression system. SDS-PAGE analysis showed the enzyme to be extremely pure and isoelectric focusing gel electrophoresis showed that the enzyme has an isoelectric point of 7.5. Immunoaffinity-purified recombinant human asparagine synthetase demonstrated both glutamine-dependent and ammonia-dependent asparagine synthetase activities, as well as glutaminase activity.
Sheng
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Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville 32610.
Moraga
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DA
Van Heeke
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G
Schuster
S M
SM
D000911:Antibodies, Monoclonal; D001217:Aspartate-Ammonia Ligase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D003429:Cross Reactions; D004790:Enzyme Induction; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006825:Hybridomas; D011401:Promoter Regions, Genetic / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D001217:Aspartate-Ammonia Ligase