A novel method for the purification of recombinant subunit I of the Dolichos biflorus seed lectin.
Lectins are carbohydrate-binding proteins that are ubiquitous in nature. Their ability to specifically bind carbohydrates has been used as a means of purification mainly through affinity chromatography techniques. Plant lectins are one of the most thoroughly studied class of lectins, however, details of their in situ function remains elusive. Recent advances in recombinant DNA techniques have been used in several laboratories to study the function of these lectins by heterologous overexpression. The larger subunit of the Dolichos biflorus seed lectin was described by Chao et al. in 1994 and purification through affinity chromatography techniques was described. Here we report on a new method for the purification of this recombinant protein with techniques that are not dependent on the ability of the lectin to bind sugars. This method may have uses in the purification of mutant proteins that may not bind carbohydrates. Characterization of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization (MALDI) mass spectroscopy shows that the lectin is over 99% pure with a molecular weight of 27,090 +/- 16.17 Da, and hemagglutination assays confirm that the lectin retains its biological activity.
Deb
S
S
School of Pharmacy and Biomedical Sciences, University of Portsmouth, UK.
Manfield
I
I
Carpenter
B
B
D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D037102:Lectins / Q000302:isolation & purification*; D037121:Plant Lectins; D011994:Recombinant Proteins / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
C086819:DB58 lectin, Dolichos biflorus; D037102:Lectins; D037121:Plant Lectins; D011994:Recombinant Proteins