EXPRESSION AND PARTIAL CHARACTERIZATION OF DOLICHOS-BIFLORUS SEED LECTIN IN ESCHERICHIA-COLI

The seed lectin from the legume, Dolichos biflorus, was expressed in E scherichia coli using the pET expression vector. Replacement of the 22 -amino acid signal sequence of this lectin with a methionine increased the level of lectin expression greater than 100-fold. Approximately 2 0% of the expressed seed lectin was soluble; the remainder was solubil ized in 8 M urea and renatured by rapid dilution. No difference in phy sicochemical properties or activity was detected between the soluble a nd renatured forms. NH2-terminal amino acid analysis and immunoblots, using antibodies that recognize the COOH-terminus of only the nontrunc ated subunit of the native heteroligomer, established that the express ed lectin has a primary structure equivalent to subunit I of the nativ e seed lectin. The expressed seed lectin is active as evidenced by its ability to bind to blood group A + H substance-Sepharose and to be sp ecifically eluted from this column with N-acetylgalactosamine. However , a comparison of the activity of the expressed lectin with the native seed lectin using a sensitive ELISA showed that the expressed lectin has a slightly lower affinity for blood group A + H substance than the native seed lectin. The expressed lectin also has a lower M(r) than t he seed lectin as determined by molecular exclusion chromatography. (C ) 1994 Academic Press, Inc.