Many proteins are still routinely expressed prokaryotically in Escherichia coli, some because they are toxic to eukaryotes. Immunotoxins, which are fusion proteins of a targeting moiety and a truncated Pseudomonas exotoxin A, kill target cells by arresting protein synthesis. Thus, immunotoxins must be expressed in E. coli. Proteins expressed in E. coli are contaminated by endotoxin (also called lipopolysaccharides (LPS)). LPS binds to toll-like receptors, inducing up to life-threatening systemic inflammation in mammals. Therefore, accepted LPS limits for therapeutics as well as for substances used in immunological studies in animals are very low. Here, we report the use of Triton X-114 and polyamine-based wash strategies, which only in combination achieved LPS-contamination well below FDA limits. Resulting LPS-reduced immunotoxins were purer and up to 2.4-fold more active in vitro. Increased activity was associated with a 2.4-fold increase in affinity on cell surface expressed target antigen. The combination method maintained enzymatic function, protein stability, and in vivo efficacy and was effective for Fab as well as dsFv formats. With some modifications, the principle of this novel combination may be applied to any chromatography-based purification process.

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Immunotoxins, which are fusion proteins of an antibody fragment and a fragment of a bacterial or a plant toxin, induce apoptosis in target cells by inhibition of protein synthesis. ADP-ribosylating toxins often have few Lysine residues in their catalytic domain. As they are the target for ubiquitination, the low number of Lysines possibly prevents ubiquitin-dependent degradation of the toxin in the cytosol. To reduce this potential degradation, we aimed to generate a Lysine-free (noK), Pseudomonas exotoxin (PE)-based immunotoxin. The new generation 24 kDa PE, which lacks all but the furin-cleavage site of domain II, was mutated at Lysine 590 (K590) and at K606 in a CD22-targeting immunotoxin and activity was determined against various B cell malignancies in vitro and in vivo. On average, K590 mutated to Arginine (R) reduced cytotoxicity by 1.3-fold and K606R enhanced cytotoxicity by 1.3-fold compared to wild type (wt). Mutating K590 to Histidine or deleting K590 did not prevent this loss in cytotoxicity. Neither stability nor internalization rate of K590R could explain reduced cytotoxicity. These results highlight relevance of Lysine 590 for PE intoxication. In line with in vitro results, the K606R mutant was more than 1.8-fold more active than the other variants in vivo suggesting that this single mutation may be beneficial when targeting CD22-positive malignancies. Finally, reduced cytotoxicity by K590R was compensated for by K606R and the resulting Lysine-free variant achieved wt-like activity in vitro and in vivo. Thus, PE24-noK may represent a promising candidate for down-stream applications that would interfere with Lysines.

Zusammenfassung: Haben Remittances, also der Geldtransfer von Migrantinnen und Migranten zur Unterstützung der Familien im Heimatland, einen positiven Einfluss auf den Wohlstand eines Landes? Hierzu können die empirischen Befunde wie folgt zusammengefasst werden: Bei den durchgeführten Paneldatenregressionen von Remittances pro Person auf das Bruttoinlandsprodukt pro Einwohner, wobei die Kontrollvariablen Arbeitslosigkeit, Export, ausländische Direktinvestitionen, Bruttoinvestitionen sowie der Einfluss der Finanzkrise 2008–2009 berücksichtigt wurden, ist der Koeffizient der Variablen Remittances pro Person mit einer Höhe von 0,026 statistisch hochsignifikant. Remittances haben demnach einen positiven Einfluss auf den Wohlstand eines Landes, wenn dieser in Bruttoinlandsprodukt pro Einwohner gemessen wird. Auch die Ergebnisse der Robustheitsanalysen haben den positiven Zusammenhang bestätigt, der auch bei Veränderung von Kontrollvariablen statistisch signifikant bleibt. Summary: Do remittances, that is, the transfer of money from migrants to support families in their home country, have a positive influence on the prosperity of a country? The empirical findings can be summarized as follows: In the panel data regression of remittances per person to the gross domestic product per inhabitant, whereby the control variables unemployment, export, foreign direct investment, gross investment and the influence of the financial crisis 2008–2009 were taken into account, the coefficient of the variable remittances per person is statistically highly significant at 0.026. Thus, remittances have a positive influence on a country’s prosperity when measured in gross domestic product per inhabitant. The results of the robustness analyses also confirmed the positive correlation, which remains statistically significant even if control variables are changed.