Mapping Domains in Proteins: Dissection and Expression ofEscherichia coliAdenylyl Cyclase

We have used the pRE expression vector containing theEscherichia coliadenylyl cyclase gene (cya) with the uniqueNdeI restriction site CATATG at the initiation codon in conjunction with a family of self-complimentary oligonucleotides to create amino- and carboxyl-terminal domains in adenylyl cyclase. The three sets of oligonucleotides contain a TAA translation stop codon in all reading frames flanked by theNdeI restriction endonuclease sequence and one or two nucleotides (5′ NNCATATGTTAATTAATTAACATATGNN 3′). Ligation of one of these annealed oligonucleotides into a restriction site or creation of 5′ TAA/CATATG 3′ translation stop/NdeI restriction site along a gene in the pRE expression vector facilitates the premature termination of protein synthesis thus yielding amino-terminal domains. Removal of a fragment of the gene corresponding to the amino-terminal portion byNdeI restriction and ligation brings the 3′ end of the gene in frame with the initiator ATG. With this strategy, expression of the carboxyl-terminal domain of a protein is possible which is otherwise not as simple as the expression of the amino-terminal domain. The feasibility of expression of any domain of a protein is demonstrated using thecyagene to create several amino- and carboxyl-terminal domains of adenylyl cyclase.