Cloning and expression of three abrin A-chains and their mutants derived by site-specific mutagenesis in Escherichia coli.

DNAs encoding of three abrin A-chains were obtained from the cDNA library of Abrus precatorius by polymerase chain reaction and ligated into the expression vector, pGEX-2T. The mature A-chains of abrins a, b and d have been expressed in the cytoplasm of Escherichia coli, and the yield of the soluble recombinant proteins was 7 mg/l induced culture. Three recombinant abrin A-chains were purified to be homogeneity and their N-glycosidase ability to inhibit protein biosynthesis in a cell-free system and to depurinate 28S rRNA in rat liver ribosomes was demonstrated in vitro. The recombinant abrin-a A-chain had the highest N-glycosidase activity among three recombinant abrin A-chains while the recombinant abrin-b A-chain, the least. Three mutants, glutamic-acid-to-alanine replacement (E164A), arginine to leucine (R167L) or double mutation (E164A and R167L) were constructed and expressed. The protein-biosynthesis-inhibitory activity of mutant (E164A), mutant (R167L) and the double mutant was found to be 25-fold, 625-fold and 1250-fold lower than that of wild type, respectively. The results indicated that Arg167 was essential for abrin toxin A-chain catalysis.