Abstract
Wrightia tinctoria globulin (WTG), one of the major seed storage proteins, was isolated for the first time from seeds of the medicinal plant. WTG was extracted and purified to homogeneity in two steps using anion-exchange and size-exclusion chromatographies. On an SDS–PAGE gel under non-reducing conditions, a major band of ~56 kDa was observed; under reducing conditions, however, two major polypeptides, one with molecular weight ~32-34 kDa and the other with molecular weight ~22-26 kDa were observed. Intact mass determination by MALDI-TOF supported this observation. The N-terminal amino acid sequence of WTG matched in NCBI database with an expressed sequence tag obtained from the c-DNA of developing embryo m-RNA of Wrightia tinctoria. The EST sequence was further substantiated by partial de novo internal sequencing using MALDI-TOF/TOF. The high sequence homology with seed storage protein 11S globulin confirmed that WTG is a type of 11S globulin. Circular dichroism analysis showed that the secondary structure of WTG consists predominantly of β-sheets (44.2%) and moderate content of α-helices (10.3%). WTG showed hemagglutinating property indicating that the protein may possess lectin-like activity. WTG was crystallized at 20 °C by the vapour diffusion method using PEG 400 as precipitant. The crystals belonged to the orthorhombic space group P212121 with cell dimensions of a=109.9Å, b=113.2Å and c=202.2Å with six molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.2Å under cryocondition. Preliminary structure solution of WTG indicated the possibility of a hexameric assembly in its asymmetric unit.
Keywords: Apocynaceae, hemagglutinating activity, Wrightia tinctoria, 11S globulin
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