Independent domain folding of Pseudomonas exotoxin and single-chain immunotoxins: Influence of interdomain connections

Ulrich Brinkmann, Johannes Buchner, Ira Pastan

Research output: Contribution to journalArticlepeer-review

98 Scopus citations


We have studied the refolding of completely unfolded and reduced Pseudomonas exotoxin (PE) and of recombinant single-chain immunotoxins made with monoclonal antibody B3 that are composed of a heavy-chain variable rigion connected by a flexible linker to the corresponding light-chain variable region (Fv), which is in turn fused to a truncted form of PE. We have found by direct activity assays that different functional domains of these multifunctional proteins fold independently with different kinetics. The ADP-ribosylation domain of PE and of the recombinant immunotoxin rapidly, whereas the assembly of the binding and/or translocation domains is regained more slowly. The complete refolding of native PE occurs more rapidly than the refolding of the recombinant immunotoxins. To determine the influence of the connector region between the B3(Fv) moiety and the toxin on the folding process of the recombinant immunotoxin B3(Fv)-PE38KDEL, we have made two different mutations in the peptide that connects the single-chain Fv domain to domain II to PE. These molecules show different folding kinetics, differences in their propensity to aggregate, and different yields of correctly folded molecules. A mutation that decreases aggration increases the rate of formation and the yield of active immunotoxin molecules.

Original languageEnglish
Pages (from-to)3075-3079
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number7
StatePublished - 1992
Externally publishedYes


  • B3(Fv)-PE38KDEL
  • Protein engineering
  • Renaturation


Dive into the research topics of 'Independent domain folding of Pseudomonas exotoxin and single-chain immunotoxins: Influence of interdomain connections'. Together they form a unique fingerprint.

Cite this