Generation of a soluble and stable apoptin-EGF fusion protein, a targeted viral protein applicable for tumor therapy

Abstract

A promising candidate for tumor targeted toxins is the chicken anemia-derived protein apoptin that induces tumor-specific apoptosis. It was aimed to design a novel apoptin-based targeted toxin by genetic fusion of apoptin with the tumor-directed ligand epidermal growth factor (EGF) using Escherichia coli as expression host. However, apoptin is highly hydrophobic and tends to form insoluble aggregates. Therefore, three different apoptin-EGF variants were generated. The fusion protein hexa-histidine (His)-apoptin-EGF (HAE) was expressed in E. coli and purified under denaturing conditions due to inclusion bodies. The protein solubility was improved by maltose-binding protein (MBP) or glutathione S-transferase. The protein MBP-apoptin-EGF^His (MAEH) was found favorable as a targeted toxin regarding final yield (4-6 mg/L) and stability. MBP was enzymatically removed using clotting factor Xa, which resulted in low yield and poor separation. MAEH was tested on target and non-target cell lines. The targeted tumor cell line A431 showed significant toxicity with an IC₅₀ of 69.55 nM upon incubation with MAEH while fibroblasts and target receptor-free cells remained unaffected. Here we designed a novel EGF receptor targeting drug with high yield, purity and stability.

Keywords: Apoptin; Apoptosis; Chicken anemia virus; Epidermal growth factor receptor; Inclusion bodies; Recombinant fusion toxin; Targeted tumor therapy.