Production of pea lectin in Escherichia coli.
In order to explore the molecular basis for the glycopeptide specificity of legume lectins, we have developed an experimental system in which specific amino acid alterations can be introduced into the carbohydrate binding site of pea lectin. This system is based on the production of pea lectin in Escherichia coli. The plasmid coding for the lectin was constructed from two lectin cDNA sequences isolated from Pisum sativum seeds (Higgins, T. J. V., Chandler, P. M., Zurawski, G., Button, S. C., and Spencer, D. (1983) J. Biol. Chem. 258, 9544-9549) and an expression vector based on the gene for the outer membrane lipoprotein of E. coli (Nakamura, K., and Inouye, M. (1982) EMBO J. 1, 771-775). The lectin is produced as a single polypeptide chain and forms insoluble aggregates in E. coli cells (2-5 mg/liter). Functional lectin is recovered by solubilization of the aggregates in guanidinium hydrochloride, renaturation in the presence of MnCl2 and CaCl2, and affinity purification on Sephadex. This procedure yields a homogeneous 28,000-dalton protein. Comparison of the recombinant lectin with natural pea lectin in an inhibition of hemagglutination assay demonstrated that there is no detectable difference in the carbohydrate binding properties of the two lectins.
D000595:Amino Acid Sequence; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002712:Chlorides; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D006386:Hemagglutination Tests; D037102:Lectins / Q000096:biosynthesis*; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D015636:Magnesium Chloride; D008345:Manganese / Q000378:metabolism; D017895:Manganese Compounds; D008970:Molecular Weight; D037121:Plant Lectins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D002712:Chlorides; D037102:Lectins; D046911:Macromolecular Substances; D017895:Manganese Compounds; D037121:Plant Lectins; C036292:pea lectin; D015636:Magnesium Chloride; D008345:Manganese; D004247:DNA; D008274:Magnesium; C025340:manganese chloride