Characterization of a β‐galactosidase hybrid protein carrying the catalytic domain of Escherichia coli adenylate cyclase

A hybrid protein of Escherichia coli , exhibiting both adenylate cyclase and β‐galactosidase activities, was purified and characterized. This protein, obteined by genetic engineering, contained the first 556 amino acids of adenylate cyclase connected to the eighth‐residue of β‐galactosidase through a pentapeptide Val‐Gly‐Asp‐Pro‐Val. The fusion protein was less stable than the native β‐galactosidase. Trypsin cleaved preferentially the adenylate cyclase moiety of the hybrid protein at a ratio of 1/50 (w/w). The kinetic properties of the hybrid protein were comparable, with a few exceptions, to those of native adenylate cyclase and β‐galactosidase. ‘Truncated’ adenylate cyclase was no longer sensitive to inhibition by excess ATP, which seems to indicate a second nucleotide binding site of wild‐type adenylate cyclase. Photoirradiation of the hybrid protein with 8‐azidoadenosine 5′‐triphosphate inactivated the adenylate cyclase activity, leaving intact the β‐galactosidase activity. A radiolabeled ATP analog was incorporated after photoirradiation into the adenylate cyclase moiety of the fusion protein as shown by limited digestion with trypsin.