Molecular cloning and expression of Cucumisin (Cuc m 1), a subtilisin-like protease of Cucumis melo in Escherichia coli.
Palavras-chave
Resumo
BACKGROUND
Oral allergy syndrome resulted from plant-derived foods is frequent among adults. Allergy to melon (cucumis melo) is one of the most frequent fruit allergies in Iran. Three different major allergens have been found in Cucumis melo that Cuc m 1 (cucumisin) has been identified as the major allergen of melon. Cucumisin is an alkaline serine protease that it is found as a 78kDa protein in precursor form. The aim of this study was production of recombinant Cuc m 1 in Escherichia coli (E. coli) cells and characterization of its allergenicity property.
METHODS
Production of recombinant Cuc m 1 was carried out by cDNA cloning technique into the pET32b(+) vector using specific primers designed based on cucumisin nucleotide sequence available in Genebank database, cucumisin encoding gene and directional cloning method. Cloned plasmid into E. coli TOP10 was transformed into E. coli BL21 and expression of the protein was induced by IPTG. The recombinant protein was purified via Ni-NTA affinity chromatography using histidine tag in recombinant protein. IgE binding of this protein was assessed by IgE-immunoblotting, ELISA and inhibition ELISA.
RESULTS
The directional cloning was resulted in expression of a fusion Cuc m 1. Immunoblotting with sera of patients allergic to melon showed strong reactivity with purified protein band. Inhibition assays demonstrated that purified rCuc m 1 could be the same with natural form of Cuc m 1 in total extract.
CONCLUSIONS
In the present study, we have provided a functional recombinant cucumisin allergen, rCuc m 1 with 86kDa, which may be used as a standard allergen for clinical diagnosis and study of allergy to melon.
