Document Type : Original Article
Authors
1
Students Research Committee, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
2
Department of Medical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
3
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.
4
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, P.O. Box 71468-64685, Shiraz, Iran
10.30476/tips.2026.112987.1367
Abstract
Age-related Macular Degeneration (AMD) heavily relies on anti-VEGF therapies, but frequent injections and resistance demand novel approaches. Recombinant immunotoxins present a promising alternative by directly targeting and eliminating pathologically active cells. This study aims to design, clone, and evaluate the expression of a novel anti-VEGF-MAP30 immunotoxin in Escherichia coli. The sequence of Ranibizumab (DrugBank: DB01270) was fused to the MAP30 sequence. Following codon optimization, the 1373 bp construct was synthesized and cloned into the pET-28a(+) expression vector. The recombinant plasmid was transformed into five E. coli strains (BL21(DE3), DH5α, SHuffle T7, XL1-Blue, and Rosetta-gami). Expression was induced using 1 mM IPTG and evaluated via SDS-PAGE across different time intervals (1 to 4 hours post-induction). Sequencing and gel electrophoresis confirmed the successful cloning of the gene. Among the evaluated hosts, E. coli BL21(DE3) exhibited the highest capacity for recombinant protein production, presenting a distinct band at 131 kDa on SDS-PAGE, whereas other strains demonstrated lower expression levels. The successful design and optimal expression of the anti-VEGF-MAP30 immunotoxin in E. coli BL21(DE3) provides a foundational step toward developing targeted therapies for ocular angiogenesis and solid tumors. Future purification and in vitro assays are warranted to validate its biological efficacy.
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