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Protein Engineering vol. 3 no. 8 pp. 739-744, 1990
© 1990 Oxford University Press

RESEARCH-ARTICLE

Expression of human asparagine synthetase in Saccharomyces cerevisiae

Gino van Heeke1 and Sheldon M. Schuster2,3

Department of Chemistry, University of Nebraska Lincoln, FL 32610, USA 2Department of Biochemistry and Molecular Biology, College of Medicine, Box J-245, University of Florida Gainesville, FL 32610, USA

3To whom correspondence should be addressed

Human asparagine synthetase was expressed in the yeast Saccharomyces cerevisiae. The identity of the expressed protein was confirmed by immunoblotting and in vitro enzymatic activity. The recombinant enzyme was shown to have both the ammonia-and glutamine-dependent asparagine synthetase activity in vitro. In contrast to overproduction in Escherichia coli, the expressed protein was found to be soluble in the yeast cell. Furthermore, expression in yeast made it possible to isolate non-degraded human asparagine synthetase which had also the N-terminal methionine correctly processed. The yeast expression plasmid was constructed for optimal production of the recombinant enzyme. In addition, unique restriction enzyme sites that bracket the first five codons of the human asparagine synthetase gene were introduced. This will allow the use of oligonucleotide cassette mutagenesis to investigate the role of the N-terminal amino acids in asparagine synthetase enzymatic activity.

Keywords: Saccharomyces cerevisiae/asparagine synthetase/N-terminal methionine/oligonucleotide cassette mutagenesis

Received November 15, 1989;


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