An efficient method for the purification and quantification of a galactose-specific lectin from vegetative tissues of Dolichos lablab.
The affinity purified galactose-specific seed lectin from Dolichos lablab, designated as DLL-II, is a tetrameric protein with an apparent native molecular mass of 120 kDa that is composed of two non-identical subunits of 31 and 29 kDa, respectively, associated non-covalently. The stems and leaves of the D. lablab plant also contain a galactose-specific lectin that cross-reacts with the seed lectin antiserum (antiserum raised against the 31 kDa subunit of DLL-II). Anti-lectin antibodies have been purified from this antiserum using a gel containing purified DLL-II lectin. Lectin specific antibodies have been used to develop simple and efficient immuno-affinity matrix, which allowed the purification of the lectin from stems and leaves of the D. lablab. The vegetative lectin (DLL-VL) exhibits similar electrophoretic properties as the seed lectin. Using these antibodies, an ELISA method was developed that allowed quantification of the lectin in the vegetative tissues (stems, leaves and roots) at concentrations of 0.5-50 ng. MS and database analysis of the tryptic peptides of the purified subunits of the DLL-VL suggested the purified protein to be a lectin.
Rameshwaram
Nagender Rao
NR
Protein Biochemistry and Molecular Biology Laboratory, University of Hyderabad, Hyderabad 500 046, India.
Nadimpalli
Siva Kumar
SK
D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods; D031294:Dolichos / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005690:Galactose / Q000032:analysis* / Q000302:isolation & purification; D018515:Plant Leaves / Q000737:chemistry; D037121:Plant Lectins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D018547:Plant Stems / Q000737:chemistry; D015203:Reproducibility of Results; D012639:Seeds / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D000906:Antibodies; D037121:Plant Lectins; D005690:Galactose