The aspartate transcarbamylase domain of a mammalian multifunctional protein expressed as an independent enzyme in Escherichia coli (original) (raw)
Summary
Although aspartate transcarbamylase (ATCase) is an independent, monofunctional enzyme in Escherichia coli, mammalian ATCase is one of the globular enzymatic domains of the multifunctional CAD protein. We subcloned fragments of the hamster CAD cDNA and assayed polypeptide products expressed in E. coli for ATCase activity in order to isolate a stretch of cDNA which encodes only the ATCase domain. Three such expression constructs contain fragments of hamster CAD cDNA similar in length to the gene encoding the E. coli ATCase catalytic subunit (pyrB). These constructs yield stable proteins with ATCase activity, ascertained by both in vivo and in vitro assays; the clones also possess sequence homology with the pyrB gene at both the 5′ and 3′ ends. The clone producing the most active ATCase contains cDNA which is analogous to the entire pyrB gene, plus a small amount of CAD sequence upstream of this region. Because these constructs produce independently folded, active ATCase from a piece of cDNA the size of the E. coli pyrB gene, they open the door for the in-depth investigation of the isolated mammalian enzyme domain utilizing recombinant DNA technology. This approach is potentially useful for the analysis of domains of other multifunctional proteins.
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Abbreviations
(EC 2.1.3.2):
ATCase, aspartate transcarbamylase
CAD:
the trifunctional protein catalyzing the first three steps of pyrimidine biosynthesis in higher eukaryotes
(EC 6.3.5.5):
CPSaseII, glutamine-dependent carbamylphosphate synthetase II
(EC 3.5.2.3):
DHOase, dihydroorotase
IPTG:
isopropyl-β-d-thiogalactopyranoside
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Authors and Affiliations
- Department of Microbiology and Immunology, Albert B. Chandler Medical Center, University of Kentucky, 40536-0084, Lexington, KY, USA
Julie A. Maley & Jeffrey N. Davidson
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- Julie A. Maley
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Communicated by B.H. Judd
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Maley, J.A., Davidson, J.N. The aspartate transcarbamylase domain of a mammalian multifunctional protein expressed as an independent enzyme in Escherichia coli.Mol Gen Genet 213, 278–284 (1988). https://doi.org/10.1007/BF00339592
- Received: 25 February 1988
- Issue Date: August 1988
- DOI: https://doi.org/10.1007/BF00339592