Hung C H, Lee M C, Chen J K, Lin J Y
Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Republic of China.
Eur J Biochem. 1994 Jan 15;219(1-2):83-7. doi: 10.1111/j.1432-1033.1994.tb19917.x.
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.
通过聚合酶链反应从鸡母珠的cDNA文库中获得了编码三种相思子毒素A链的DNA，并将其连接到表达载体pGEX-2T中。相思子毒素a、b和d的成熟A链已在大肠杆菌细胞质中表达，可溶性重组蛋白的产量为7 mg/l诱导培养物。三种重组相思子毒素A链被纯化至同质，并在体外证明了它们在无细胞系统中抑制蛋白质生物合成以及使大鼠肝脏核糖体中的28S rRNA脱嘌呤的N-糖苷酶能力。重组相思子毒素-a A链在三种重组相思子毒素A链中具有最高的N-糖苷酶活性，而重组相思子毒素-b A链的活性最低。构建并表达了三个突变体，即谷氨酸到丙氨酸替换（E164A）、精氨酸到亮氨酸（R167L）或双突变（E164A和R167L）。发现突变体（E164A）、突变体（R167L）和双突变体的蛋白质生物合成抑制活性分别比野生型低25倍、625倍和1250倍。结果表明，Arg167对相思子毒素A链催化至关重要。
