Cloning and Expression of Three Abrin A-Chains and Their Mutants Derived by Site-Specific Mutagenesis in Escherichia coli

10.30047/JGMB.199412.0008

Chih-Hung Hung；Ming-Chih Lee；Jeen-Kuan Chen；Jung-Yaw Lin

Journal of Genetics and Molecular Biology

5卷4期（1994 / 12 / 01）

77 - 77

英文

DNAs encoding of three abrin A-chains were obtained from the cDNA library of Abrus precatorius by PCR and ligated into the expression vector, pGEX-2T. The mature A-chains of abrin-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 of induced culture. Three reabrin A-chains were purified to be homogenous and their N-glycosidase activity to inhibit the protein biosynthesis of cell free system and to depurinate 28S rRNA in rat liver ribosomes was demonstrated in vitro. The reabrin-a A-chainhad the highest N-glycosidase activity among three reabrin A-chains while the reabrin-b A-chai, the lease. 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, 625 and 1250-fold lower than that of wile type, respectively. The results indicated that Arg167 was essential for abrin toxin A-chain catalysis.