Expression and partial characterization of Dolichos biflorus seed lectin in Escherichia coli.
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.
Chao
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Q
Section of Molecular and Cellular Biology, University of California, Davis 95616.
Casalongue
C
C
Quinn
J M
JM
Etzler
M E
ME
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
D002241:Carbohydrates; D017931:DNA Primers; D037102:Lectins; D037121:Plant Lectins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C036554:dolichos biflorus agglutinin