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J. Biol. Chem., Vol. 267, Issue 4, 2244-2250, Feb, 1992
D Ladant, P Glaser and A Ullmann
We developed an improved method of linker insertion mutagenesis for
introducing 2 or 16 codons into the Bordetella pertussis cyaA gene which
encodes a calmodulin-dependent adenylate cyclase. A recombinant kanamycin
resistance cassette, containing oligonucleotide linkers, was cloned in
plasmids which carried a truncated cyaA gene, fused at its 3' end to the 5'
end of the Escherichia coli lacZ gene, specifying the alpha-peptide. This
construction permitted a double selection for in- frame insertions by using
screening for kanamycin resistance and for lactose-positive phenotype,
resulting from alpha-complementation. We showed that most of the two-amino
acid insertions within the N-terminal moiety of the catalytic domain of
adenylate cyclase abolished enzymatic activity and/or altered the stability
of the protein. All two-amino acid insertions within the C-terminal part of
adenylate cyclase resulted in fully stable and active enzymes. These
results confirm the modular structure of the catalytic domain of adenylate
cyclase, previously proposed on the basis of proteolytic studies. Two-amino
acid insertions between residues 247-248 and 335-336 were shown to affect
the calmodulin responsiveness of adenylate cyclase, suggesting that the
corresponding region in the enzyme is involved in the binding of calmodulin
or in the process of calmodulin activation. In addition, we have identified
within the primary structure of adenylate cyclase several permissive sites
which tolerate 16-amino acid insertions without interfering with the
catalytic activity or calmodulin binding. By inserting foreign antigenic
determinants into these permissive sites the resulting recombinant
adenylate cyclase toxin could be used to deliver specific epitopes into
antigen-presenting cells.
Insertional mutagenesis of Bordetella pertussis adenylate cyclase
Unite de Biochimie des Regulations Cellulaires, Institut Pasteur, Paris, France.
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