title: Expression and partial characterization of Dolichos biflorus seed lectin in Escherichia coli.
abstract: The seed lectin from the legume, Dolichos biflorus, was expressed in Escherichia 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 20% of the expressed seed lectin was soluble; the remainder was solubilized in 8 M urea and renatured by rapid dilution. No difference in physicochemical properties or activity was detected between the soluble and renatured forms. NH2-terminal amino acid analysis and immunoblots, using antibodies that recognize the COOH-terminus of only the nontruncated subunit of the native heteroligomer, established that the expressed lectin has a primary structure equivalent to subunit I of the native 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 specifically 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 had a lower M(r) than the seed lectin as determined by molecular exclusion chromatography.
authors.affiliation: Section of Molecular and Cellular Biology, University of California, Davis 95616.
authors.forename: Q
authors.initials: Q
authors.lastname: Chao
authors.forename: C
authors.initials: C
authors.lastname: Casalongue
authors.forename: J M
authors.initials: JM
authors.lastname: Quinn
authors.forename: M E
authors.initials: ME
authors.lastname: Etzler
mesh_terms: D001483:Base Sequence; D002241:Carbohydrates; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004926:Escherichia coli; D015151:Immunoblotting; D037102:Lectins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D037121:Plant Lectins; D016133:Polymerase Chain Reaction / Q000379:methods; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012639:Seeds
chemical_list: D002241:Carbohydrates; D017931:DNA Primers; D037102:Lectins; D037121:Plant Lectins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C036554:dolichos biflorus agglutinin