Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Expression of recombinant human asparagine synthetase and mutational analysis of the active site
Abstract
Human asparagine synthetase was expressed in both Escherichia coli and Saccharomyces cerevisiae. Inducible, plasmid-derived expression of soluble and enzymatically active asparagine synthetase in E. coli was found to be temperature dependent. Active recombinant enzyme could be recovered only when the incubation temperature during expression was lowered to 30$\sp\circ$C or less. The enzyme exhibited both the ammonia- and glutamine-dependent AS activity in vitro. Enzymatic activity was also demonstrated in vivo by complementation studies in an asparagine auxotrophic E. coli strain. The recombinant human asparagine synthetase was purified and its degradation pattern was studied. In contrast to earlier findings, these studies suggested that the active enzyme is a homodimer. Plasmid-derived expression of asparagine synthetase in S. cerevisiae yielded active enzyme at the normal growth temperature of yeast. Furthermore, the overproduced recombinant enzyme had the N-terminal methionine correctly removed. In contrast to recovery from E. coli, purification of human asparagine synthetase from yeast consistently yielded non-degraded enzyme. The yeast expression vector was especially constructed such that future site-directed mutagenesis experiments will be highly efficient and greatly facilitated. The functional role of the N-terminal cysteine was investigated. Site-specific mutagenesis was used to substitute this residue for an alanine. While the ammonia-dependent activity remained unaffected, the glutamine-dependent activity was completely abolished, indicating that this cysteine is essential for glutamine-dependent asparagine synthetase activity.
Subject Area
Biochemistry
Recommended Citation
Van Heeke, Gino, "Expression of recombinant human asparagine synthetase and mutational analysis of the active site" (1989). ETD collection for University of Nebraska-Lincoln. AAI8918567.
https://digitalcommons.unl.edu/dissertations/AAI8918567