High-Level Expression of Human Asparagine Synthetase and Production of Monoclonal Antibodies for Enzyme Purification
Document Type
Article
Publication Date
1-1-1992
Description
In order to obtain large quantities of extremely pure human asparagine synthetase for detailed kinetic and structural studies, its gene was cloned into a 2μ plasmid (pBS24.1GAS) suitable for replication in a Saccharomyces cerevisiae cir° 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.
Citation Information
Sheng, Shijie; Moraga, David A.; Van Heeke, Gino; and Schuster, Sheldon M.. 1992. High-Level Expression of Human Asparagine Synthetase and Production of Monoclonal Antibodies for Enzyme Purification. Protein Expression and Purification. Vol.3(4). 337-346. https://doi.org/10.1016/1046-5928(92)90010-T PMID: 1358303 ISSN: 1046-5928