Linker insertion mutagenesis based on IS21 transposition: isolation of an AMP-insensitive variant of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa.

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Serval ID
serval:BIB_BEED51274039
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Linker insertion mutagenesis based on IS21 transposition: isolation of an AMP-insensitive variant of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa.
Journal
Protein Engineering
Author(s)
Seitz T., Berger B., Nguyen V.T., Tricot C., Villeret V., Schmid S., Stalon V., Haas D.
ISSN
0269-2139 (Print)
ISSN-L
0269-2139
Publication state
Published
Issued date
2000
Volume
13
Number
5
Pages
329-337
Language
english
Abstract
The bacterial insertion sequence IS21 when repeated in tandem efficiently promotes non-replicative cointegrate formation in Escherichia coli. An IS21-IS21 junction region which had been engineered to contain unique SalI and BglII sites close to the IS21 termini was not affected in the ability to form cointegrates with target plasmids. Based on this finding, a novel procedure of random linker insertion mutagenesis was devised. Suicide plasmids containing the engineered junction region (pME5 and pME6) formed cointegrates with target plasmids in an E.coli host strain expressing the IS21 transposition proteins in trans. Cointegrates were resolved in vitro by restriction with SalI or BglII and ligation; thus, insertions of four or 11 codons, respectively, were created in the target DNA, practically at random. The cloned Pseudomonas aeruginosa arcB gene encoding catabolic ornithine carbamoyltransferase was used as a target. Of 20 different four-codon insertions in arcB, 11 inactivated the enzyme. Among the remaining nine insertion mutants which retained enzyme activity, three enzyme variants had reduced affinity for the substrate ornithine and one had lost recognition of the allosteric activator AMP. The linker insertions obtained illustrate the usefulness of the method in the analysis of structure-function relationships of proteins.
Keywords
Adenosine Monophosphate/metabolism, Amino Acid Sequence, Base Sequence, DNA Primers, DNA Transposable Elements, DNA, Recombinant, Models, Molecular, Molecular Sequence Data, Mutagenesis, Insertional, Ornithine Carbamoyltransferase/chemistry, Ornithine Carbamoyltransferase/genetics, Point Mutation, Pseudomonas aeruginosa/enzymology
Pubmed
Web of science
Open Access
Yes
Create date
25/01/2008 18:01
Last modification date
25/09/2019 7:10
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