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Protein Engineering, Vol. 13, No. 5, 329-337, May 2000
© 2000 Oxford University Press

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

Thomas Seitz1, Bernard Berger1, Van Thanh Nguyen1,2, Catherine Tricot2, Vincent Villeret2, Sergio Schmid3, Victor Stalon2,4 and Dieter Haas1,5

1 Laboratoire de Biologie Microbienne, Université de Lausanne, CH-1015 Lausanne, Switzerland, 2 Institut de Recherches Microbiologiques Jean-Marie Wiame, B-1070 Brussels, Belgium, 3 Ecole d'Ingénieurs du Valais, CH-1950 Sion, Switzerland and 4 Laboratoire de Microbiologie, Université Libre de Bruxelles, B-1070 Brussels, Belgium

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.


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F. Narberhaus and S. Balsiger
Structure-Function Studies of Escherichia coli RpoH ({sigma}32) by In Vitro Linker Insertion Mutagenesis
J. Bacteriol., May 1, 2003; 185(9): 2731 - 2738.
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