Targeted expression and purification of elastin-like polypeptide-fused abrin: A potential strategy for cancer therapy

Abstract

Abrin is a potent plant-derived toxin that targets 28S rRNA in eukaryotic ribosomes through its N-glycosidase activity. Its ability to induce apoptosis makes it a promising candidate for cancer-targeted immunotoxin therapy. To enhance the expression of Abrin in E. coli, its coding sequence was optimized by replacing native codons with synonymous codons that match codon usage preferences of E. coli, applying both codon usage frequency and codon harmonization strategies. The optimized sequence was generated using a two-step PCR process and cloned into expression vectors to produce both native and optimized unfused Abrin, as well as Elastin-Like Polypeptide fused forms. These included the constructs pET. native Abrin and pET. optimized Abrin (with His-tags), as well as pET. native Abrin-ELP and pET. optimized Abrin-ELP (with ELP tags). All constructs were successfully expressed in E. coli. Codon optimization led to approximately a two-fold increase in Abrin accumulation, resulting in stable expression levels comprising 70.72 % of total soluble protein. MTT assays demonstrated a concentration-dependent antiproliferative effect of Abrin on LS180 and MCF-7 cancer cells. It is noteworthy that the ELP-fused proteins significantly increased cytotoxic activity in LS180 cells, with IC₅₀ values of 5.329 µg/mL for native Abrin-ELP and 4.828 µg/mL for the optimized Abrin-ELP. These findings highlight the therapeutic potential of ELP-fused Abrin as a targeted cancer treatment.