title: Expression of human asparagine synthetase in Escherichia coli.
abstract: Human asparagine synthetase was expressed in Escherichia coli. Synthesis of the enzyme was demonstrated by immunoblotting and by complementation of asparagine auxotrophy in E. coli. The recombinant enzyme was shown to have both the ammonia- and glutamine-dependent asparagine synthetase activity in vitro. Compared to asparagine synthetase isolated from beef pancreas, the one expressed in E. coli migrated at a slightly slower rate on a denaturing protein gel. In contrast with previous reports, the data obtained here strongly suggest that the active enzyme is a homodimer. The production of soluble and active enzyme was shown to be highly temperature-dependent. Expression at 37 degrees C yielded no soluble enzyme, whereas growth at 30 and 21 degrees C favored the production of soluble asparagine synthetase. The incubation temperature was also important for complementation of asparagine auxotrophy in E. coli, as growth in the absence of asparagine occurred at 30 degrees C and not at 37 degrees C.
authors.affiliation: Department of Chemistry, University of Nebraska, Lincoln 68588-0304.
authors.forename: G
authors.initials: G
authors.lastname: Van Heeke
authors.forename: S M
authors.initials: SM
authors.lastname: Schuster
mesh_terms: D001217:Aspartate-Ammonia Ligase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D008025:Ligases / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D008025:Ligases; D001217:Aspartate-Ammonia Ligase