Expression of Erythrina corallodendron lectin in Escherichia coli

The cDNA of the Erythrina corallodendron lectin ( ECor L) has been expressed in Escherichia coli . For this purpose, an Nco I site was inserted into the cDNA coding for the lectin precursor [Arango, R., Rozenblatt, S. & Sharon, N. (1990) FEBS Lett. 264 , 109–112] immediately before the codon GTG (103–105) which codes for the N‐terminal valine of the mature lectin. This introduced an ATG codon for a methionine preceding the valine. The mutated cDNA was ligated into pUC‐8, then subcloned into the expression vector pET‐3d, which carries a strong promoter derived from gene 10 of the phage T7. The recombinant plasmid was introduced into the E. coli lysogenic strain BL21(DE3). Recombinant ECor L was expressed by growing the bacteria in the presence of isopropyl β ‐ d ‐thiogalactopyranoside. Most of the recombinant lectin was found in an insoluble aggregated form as inclusion bodies and only a small part was in the culture medium in a soluble active form. Functional recombinant lectin was recovered from the inclusion bodies by solubilization with 6 M urea in cyclohexylaminopropane sulfonate pH 10.5, renaturation by 10‐fold dilution in the same buffer and further adjustment of the pH to 8.0. The recombinant lectin, obtained at a yield of 4–7 mg/1 culture, had, by gel filtration, a slightly lower molecular mass (56 kDa) than the native lectin, and was devoid of covalently linked carbohydrate; it was, however, essentially indistinguishable from native ECor L by other criteria, including its dimeric structure, Western blot analysis with anti‐ ECor L polyclonal and monoclonal antibodies, and Ouchterlony double‐diffusion analysis with polyclonal antibodies, as well as hemagglutinating activity and specificity for mono‐ or disaccharides.