Expression of Erythrina corallodendron lectin in Escherichia coli.
The cDNA of the Erythrina corallodendron lectin (ECorL) has been expressed in Escherichia coli. For this purpose, an NcoI 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 ECorL was expressed by growing the bacteria in the presence of isopropyl beta-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/l 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 ECorL by other criteria, including its dimeric structure, Western blot analysis with anti-ECorL polyclonal and monoclonal antibodies, and Ouchterlony double-diffusion analysis with polyclonal antibodies, as well as hemagglutinating activity and specificity for mono- or disaccharides.
Arango
R
R
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Adar
R
R
Rozenblatt
S
S
Sharon
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004895:Erythrina / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006384:Hemagglutination; D006801:Humans; D005779:Immunodiffusion; D037102:Lectins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D037121:Plant Lectins; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping
D003062:Codon; D037102:Lectins; D009838:Oligodeoxyribonucleotides; D037121:Plant Lectins; D011994:Recombinant Proteins; C045629:erythrina lectin; D004247:DNA