Study On Purification And Characterization Of Phaseolus Multiflorus. Willd Agglutinin Molecular Cloning And Analyzing Of Agglutinin From Polygonatum Odoratum (Mill.) Druce

A lectin was isolated from the seed of Phaseolus multiflorus. Willd by extraction, precipitation with (NH₄)₂SO₄, anion-exchange chromatography on DEAE-Sepharose column, cation-exchange chromatography on CM-Sepharose column followed by gel filtration on a Sephacryl S-200 column. The apparent molecular mass of P. multiflorus agglutinin (PML) was 56 KD and purified PML show a single band in SDS-PAGE with a molecular mass of 28KD. Therefore, the intact PML was considered to be a dimer glycoprotein with two identical subunits. The lectin was a potent agglutinin for rabbit erythrocytes. Glucosamine Hydrochloride was the most potent inhibitor of PML hemagglutination of rabbit erythrocytes, followed by sucrose. The fluorescence spectrum of PML excited at 280nm and 295nm showed a maximum peak at 327nm. The characteristic peak of Tyr did not exist, which showed that the fluorescence energy of Tyr was transfered to Trp and strength the fluorescence of Trp. When PML was excited at 295nm, the fluorescence spectrum showed a maximum peak at 327nm, theλmax of fluorescence emission spectrum blue-shifted 21nm compared with theλmax of free Tyr(348nm), which indicated that tryptophan residues were embedded in the protein matrix allowing a modest water accessibility or located near the surface of the molicule. The change of agglutinating activity in different temperature and pH indicated that PML had no hemagglutinating activity at 80℃. The effect of chemical modification of amino acid residues upon hemagglutination was investigated. There was a definite decrease of fluorescence intensity which corresponds to the oxidation of tryptophan. Under native conditions about 3 tryptophan residues were available for modification by NBS, which had effect on the hemagglutinating activity. Fluorescence quenching study on PML with KI and acrylamide showed that KI and acrylamide quenched the fluorescence of lectin through dynamic quenching mechanism, and 83ï¼…of Trp was quenched by acrylamide, 49ï¼…of Trp was quenched by KI. Polygonatum odoratum Agglutinin (POL) from Amaryllidaceae species is a mannose-binding protein with various biological activities. The total RNA of Polygonatum odoratum was extracted, and then reverse transcript into cDNA. A primer was designed based on the conserved regions of other MBL plant's agglutinin through homology alignment. Using theRT-PCR, 3' RACE (rapid amplification of cDNA ends) technique, a DNA fragments was first cloned from Polygonatum odoratum by performing PCR that used Polygonatum odoratum cDNA genome as the templete. Then another two primers were designed according to the sequence of 3' ends, one was used to reverse transcript the RNA into cDNA, poly A were added to the 3' ends of cDNA with the function of TdT(Terminal Deoxynucleotidyl Transferase), using the 5' RACE technique the fragment of about four hunderd nucleotides was cloned. The two partially overlapping cDNA fragments were assembled a full-length cDNA sequence of Polygonatum odoratum.The full-length cDNA had 529bp, and the sequence encoded an open reading frame of 157 amino acids. The result showed that POL gene encoded a protein precursor with a signal peptide,mature protein and C-terminal cleavage amino acids sequence by the analysis in the Blast of Genbank. The mature protein included 91 amino acids residues and the molecular weight is about 14 KD. The mature protein sequence showed the identity to those of Galanthus nivalis agglutinin, Polygonatum cyrtonema agglutinin, Polygonatum multiflorum agglutinin, Aspidistra elatior Blume agglutinin DOM1, Aspidistra elatior Blume agglutinin DOM2, Zephranthes grandiflora agglutinin respectively are 44.0ï¼…,60.9ï¼…,58.7ï¼…,43.2ï¼…,52.6ï¼…,75.9ï¼…. Blocks' analysis revealed that the deduced amino acid sequence of POE had two functional domains specific for lectin and two sugar-binding boxes (QDNY). In the mature protein, there were 39.6ï¼…hydrophobic amino acids, 42.9ï¼…hydrophilic amino acids, 9.9ï¼…basic amino acids and 7.7ï¼…acidic amino acids. The cloning of this gene established an important base to the study of POL gene structure, the cleavage mechanism, the mechanism of expression and regulation, the relationship of structure and function of POL.