Site-directed mutagenesis and bacterial expression of human adenosine deaminase

Adenosine deaminase (ADA) is a purine salvage pathway enzyme, the absence of which is associated with severe combined immunodeficiency disease. Time-resolved fluorescence studies, in the presence of enzyme inhibitors, indicate that at least one of the four tryptophans present in the protein molecule is close to (or in) the active site. To investigate the role of these tryptophan residues in enzyme function, they have cloned ADA cDNA into a vector in which expression is directed by the lambda P/sub R/ promoter. E. coli cells deficient in ADA were transformed with the vector construct and were shown to synthesize catalytically active human ADA. Site directed mutagenesis, coupled with a uracil selection technique for generating mutants with high efficiency, was used to construct mutant alleles of the cloned ADA. Eight mutants were obtained with base substitutions converting each of the four tryptophans to arginine or glycine. The correlation between these specific mutations and the functional expression of ADA has been examined in the ADA deficient bacterial host.