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Volume 132, Issue 1

Cloning and Expression of the Asparaginase Gene in and Free

Abstract

SUMMARY: A genomic library of DNA was constructed in bacteriophage λ1059 and recombinants expressing asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4·7 kb RI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from cells carrying pASN30 was indistinguishable from the enzyme on the basis of specific activity [660–700 units (mg protein)], pI value (8·5), and subunit molecular weight (32 x 10). Expression of the cloned gene was subject to glucose repression in but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into , caused increased synthesis of the enzyme (2–4 fold higher than the current production strain).

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© Society for General Microbiology 1986
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1986-01-01
2024-04-23
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Cloning and Expression of the Erwinia chrysanthemi Asparaginase Gene in Escherichia coli and Erwinia carotovora
Microbiology 132, 151 (1986); https://doi.org/10.1099/00221287-132-1-151
/content/journal/micro/10.1099/00221287-132-1-151
/content/journal/micro/10.1099/00221287-132-1-151
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