Alanine scanning mutagenesis identifies surface amino acids on domain II of Pseudomonas exotoxin required for cytotoxicity, proper folding, and secretion into periplasm.

摘要: Pseudomonas exotoxin A (PE) is a single polypeptide chain that contains 613 amino acids and arranged into three major structural domains. Domain Ia responsible for cell recognition, domain II translocation of PE across the membrane, III ADP-ribosylation elongation factor 2. Recombinant can be produced in Escherichia coli efficiently secreted periplasm when an OmpA signal sequence present. To investigate role located on surface action toxin against mammalian cells, we substituted alanine each 27 present II. Surprisingly, all mutant proteins had some alteration cytotoxicity tested human A431 or MCF7 cells mouse L929 cells. Native has compact structure therefore relatively protease resistant very little activity detected absence denaturing agents like urea dithiothreitol. Several mutations resulted altered sensitivity toxin. Seven molecules exhibited without dithiothreitol, indicating they are partially unfolded. Out these seven mutants, six increased cytotoxic at least one target lines other retained its native potency.