PRODUCTION OF AN ENZYMATICALLY INACTIVE ANALOG OF PHOSPHOLIPASE-D FROM CORYNEBACTERIUM-PSEUDOTUBERCULOSIS

The gene pld, encoding the phospholipase D (PLD) of Corynebacterium pseudotuberculosis, was mutagenized using formic acid and then expressed in Escherichia coli. Mutagenesis was targeted at the coding region of pld, so as to produce only one or a limited number of point mutations. Transformants were screened for the enzymatic and immunological properties of their PLD products. One clone was found to produce a protein which was enzymatically inactive, but which was comparable to the wild-type PLD in size and antigenicity. The sequence of the pld mutant revealed a single base change. As a consequence, the codon for His20 was converted to Tyr. These results suggest that His20 forms part of the active site of the PLD molecule. If this protein is immunogenic in sheep, it would form the basis of a genetically inactivated vaccine.