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.
Hung
C H
CH
Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Republic of China.
Lee
M C
MC
Chen
J K
JK
Lin
J Y
JY
D000036:Abrin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D015870:Gene Expression; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis / Q000187:drug effects; D012339:RNA, Ribosomal, 28S / Q000187:drug effects / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D012156:Reticulocytes / Q000378:metabolism
D017931:DNA Primers; D012339:RNA, Ribosomal, 28S; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000036:Abrin; D007930:Leucine