Expression and characterisation in E. coli of mutant forms of saporin.
In the present communication, we report on the expression and characterisation in Escherichia coli of mutant derivatives of saporin, a type 1 ribosome-inactivating protein from Saponaria officinalis L. The effects of substitution of Glu 176 with Lys and those of deletion of 19 amino acids at the C-terminal were evaluated both in vivo, testing the influence of expressed proteins on bacterial growth and in vitro measuring their N-glycosidase and supercoiled DNA relaxation activities. Results indicate that both modifications of the wild-type protein abolish its toxicity to bacterial cells and impair its enzymatic activity on polynucleotide substrates, either RNA or DNA.
Pittaluga
Eugenia
E
Department of Basic and Applied Biology, University of L'Aquila, Via Vetoio Coppito, 67100 L'Aquila, Italy.
Poma
Anna
A
Tucci
Adele
A
Spanò
Laura
L
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002474:Cell-Free System; D004278:DNA, Superhelical / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000032:analysis / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000633:toxicity*; D011994:Recombinant Proteins / Q000633:toxicity; D029750:Saponaria / Q000737:chemistry; D017384:Sequence Deletion
D004278:DNA, Superhelical; D010940:Plant Proteins; D011994:Recombinant Proteins; D009699:N-Glycosyl Hydrolases; D008239:Lysine