The modified asparaginase Was79 was derived from the recombinant wild-type L-asparaginase of Wolinella succinogenes. The Was79 contains the amino acid substitutions V23Q and K24T responsible for the resistance to trypsinolysis and the N-terminal heparin-binding peptide KRKKKGKGLGKKR responsible for the binding to heparin and tumor K562 cells in vitro. When tested on a mouse model of Fischer lymphadenosis L5178Y, therapeutic efficacy of Was79 was significantly higher than that of reference enzymes at all single therapeutic doses used (125-8000 IU/kg). At Was79 single doses of 500-8000 IU/kg, the complete remission rate of 100 % was observed. The Was79 variant can be expressed intracellularly in E. coli as a less immunogenic formyl-methionine-free form at high per cell production levels.
The anti-cancer efficacy of methionine γ-lyase (MGL) from Clostridium sporogenes (C. sporogenes) is described. MGL was active against cancer models in vitro and in vivo. The calculated EC50 values for MGL were 4.4 U/ml for A549, 7.5 U/ml for SK-BR3, 2.4 U/ml for SKOV3, and 0.4 U/ml for MCF7 cells. The combination of doxorubicin (DOX) and MGL was more effective for A549 human lung cancer growth inhibition than either agent alone in vitro and in vivo. MGL reduced the EC50 of doxorubicin from 35.9 μg/mL to 0.01-0.265 μg/mL. The growth inhibitory effect of DOX + MGL on A549 xenografts in vivo was reflective of the results obtained in vitro. The inhibition rate of tumor growth in the combined arm was 57%, significantly higher than that in the doxorubicin (p = 0.033)-alone arm.
Protein nanoparticles (NPs) can be used as vaccine platforms for target antigen presentation. Aim: To conduct a proof-of-concept study to demonstrate that an effective NP platform can be built based on a short self-assembling peptide (SAP) rather than a large self-assembling protein. Materials & methods: SUMO-based protein fusions (SFs) containing an N-terminal SAP and a C-terminal antigen were designed, expressed in Escherichia coli and purified. The structure was investigated by electron microscopy. The antibody response was tested in mice after two adjuvant-free immunizations. Results: Renatured SFs form fiber-like NPs with the antigen exposed on the surface and induce a significant antibody response with a remarkably high target-to-platform ratio. Conclusion: The platform is effective and has considerable potential for modification toward various applications, including vaccine development.
A 67-kDa protein that can specifically bind the activated Cry9A endotoxin under ligand-blotting conditions was purified from midgut epithelium apical membranes of wax moth Galleria mellonella by affinity chromatography. N-Terminal amino acid sequencing enabled identification of this protein as aminopeptidase N. In similar experiments, 66- and 58-kDa proteins specific to endotoxin Cry3A were isolated from the midgut epithelium apical membranes of Tenebrio molitor larvae. Mass spectrometry showed close similarity of the 58-kDa protein to the Tenebrio molitor α-amylase.
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