Novel fusion proteins based on recombinant lectins for delivery of a cytotoxic peptide specifically to cancer cells
type: CreativeWork
description: One of the most common post-translational modifications is covalent attachment of carbohydrates to proteins. At the cell surface, molecular recognition events involved in cancer metastasis are mediated by sugar moieties of glycoproteins. Evidence from both patient (histochemical analysis) and experimental work (tumour models) show that the metastatic tumour phenotype is associated with altered sialylation of tumour cell surfaces. Structural domains that recognize and bind specific carbohydrates without altering the recognized sugars are usually called lectins. Mistletoe lectin isoform I (MLI), from Viscum album, is an example which has strong binding affinity for sialic acids especially α2,6 sialic acid.In the present study, mistletoe lectin isoform I chain B (MLB) was targeted through conducting polymerase chain reaction (PCR) for cDNA which was reverse transcribed from the extracted V. album RNA. The targeted sequence was successfully amplified, cloned and sequenced. The sequence data showed a clear indication that the insert is our desired gene product allowing us to go on to design a fusion protein. Both N- and C- terminus fusion strategies were used to assemble constructs of the MLB chain fused to GFP in the pGFPuv expression vector which was transformed into BL21 E. coli and the expression of the target gene/fusion was successfully carried out. The same mechanism was used to generate positive and negative control fusion proteins from elderberry S. nigra lectin and snowdrop G. nivalis lectin respectively. The expressed fusions proteins were purified through affinity chromatography using CNBR-activated Sepharose 4B column coupled with degalactosylated fetuin so as to confirm the sialic acid binding activity of MLB. The binding of MLB+GFP fusion proteins to the membrane glycans of human cancer cells was also confirmed through the treatment of human metastatic melanoma cells with the fusion protein and the fusions were detected to actively attach and cross the cel…
abstract: One of the most common post-translational modifications is covalent attachment of carbohydrates to proteins. At the cell surface, molecular recognition events involved in cancer metastasis are mediated by sugar moieties of glycoproteins. Evidence from both patient (histochemical analysis) and experimental work (tumour models) show that the metastatic tumour phenotype is associated with altered sialylation of tumour cell surfaces. Structural domains that recognize and bind specific carbohydrates without altering the recognized sugars are usually called lectins. Mistletoe lectin isoform I (MLI), from Viscum album, is an example which has strong binding affinity for sialic acids especially α2,6 sialic acid.In the present study, mistletoe lectin isoform I chain B (MLB) was targeted through conducting polymerase chain reaction (PCR) for cDNA which was reverse transcribed from the extracted V. album RNA. The targeted sequence was successfully amplified, cloned and sequenced. The sequence data showed a clear indication that the insert is our desired gene product allowing us to go on to design a fusion protein. Both N- and C- terminus fusion strategies were used to assemble constructs of the MLB chain fused to GFP in the pGFPuv expression vector which was transformed into BL21 E. coli and the expression of the target gene/fusion was successfully carried out. The same mechanism was used to generate positive and negative control fusion proteins from elderberry S. nigra lectin and snowdrop G. nivalis lectin respectively. The expressed fusions proteins were purified through affinity chromatography using CNBR-activated Sepharose 4B column coupled with degalactosylated fetuin so as to confirm the sialic acid binding activity of MLB. The binding of MLB+GFP fusion proteins to the membrane glycans of human cancer cells was also confirmed through the treatment of human metastatic melanoma cells with the fusion protein and the fusions were detected to actively attach and cross the cel…
author: Mohammed, Soran
language: en
date published: 2026
date modified: 2026-07-24T04:26:41Z
provider: U. of Salford
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: U. of Salford