Optimization of extraction method of the cell wall protein of Lycium barbarum L.

【Objective】 To explore the suitable extraction scheme of cell wall protein from Lycium barbarum L. fruit, which provided technical support for the subsequent proteomic research of L. barbarum L.【Method】 Using L. barbarum L. fruit as experimental material and six-step elution method was used to remove cytoplasm proteins, then comparing cell wall protein extraction effect with calcium chloride extraction method, lithium chloride extraction method, calcium chloride plus lithium chloride extraction method and phenol extraction method by protein quantitation and electrophoresis. Cell wall proteins were examined by two dimensional electrophoresis(2-DE)and liquid chromatography(LC).【Result】 Cytoplasmic protein and other interfering substances could be removed better using the six-step elution method. The extraction effect of the four methods on cell wall protein of L. barbarum L. fruit was ranked as follows:calcium chloride extraction method(272 μg/g) >phenol extraction method(261 μg/g) >calcium chloride plus lithium chloride extraction method(217 μg/g) >lithium chloride extraction method(176 μg/g). The dissolution effect of lysis buffer I【7 mol/L urea, 2 mol/L thiourea, 30 g/L 3-【3-(cholamidopropyl)dimethylamino】 -1-propanesulfonate(CHAPS), 50 g/L dithiothreitol (DTT)】 was better, the quantitative concentration of protein was higher, and the electrophoretic bands were more and clearer. The results of two-dimensional electrophoresis of the protein samples extracted by calcium chloride were better, with more protein spots, higher clarity and resolution, and the liquid chromatography showed that there were many kinds of cell wall protein peptides extracted by calcium chloride method, and most of them had high relative abundance, and contained low abundance proteins. In the classification of the tested proteins, some proteins located on the cell wall.【Conclusion】 The method of extracting protein sample by calcium chloride alone is the best method to extract cell wall proteins from Ningxia L. barbarum L. fruit, and the obtained protein sample has reached the standard of proteomics research.