Tea Leaves, Sugar, Protein Purification Study

Glycoprotein is one of biomacromolecules that play important roles in the bioinformation and the structure in organism. Additionally,many glycoproteins from natural plants are in themselves effective medications in depressing blood-glucose,enhancing immunological function,combating inflammation and repelling tumor etc. Glycotechnology drugs have already been applied in clinic medicine. Glycoproteins from the leaves of Camellia Sinensis,are primary bioactive components of tea polysaccharide,which have the generally known effect on curing diabetes.Glycoproteins,in this paper,were extracted,isolated,detected and purified from the leaves of Camellia Sinensis. Experiments were done on extracting total proteins by using different buffer varieties and different pH. Total proteins were finally extracted with the Buffer A by the method of using the acetone powder plus a suitable amount of PVPP.Classification of protein was precipitated from proteinic extraction solutionwith different ammonium sulfate fraction--0 -20,20 -40,40 -60,60 -80.Afterwards,each classified protein was purified by Sephadex G-100 column chromatography so as to make proteins eluted according to their molecular weights. Glycoproteins were detected from the elution of classified proteins by gel chromatography.Glycoproteins from the leaves of Camellia Sinensis were respectively detected by preparatory quality of gel chromatography and Glycoprotein Staining in SDS-gel. According to the result showed at 280nm and at 490nm,in the comparison of whether protein absorption top and sugar quantity top overlapped,glycoproteins would be detected preparatorily,and as a result,tubes in two distinct areas had glycoproteins by this method. Proteins were precipitated with trichloroaceticacid and with cold acetone,and glycoprotein was determined from SDS-gel. By using this method,multi- glycoproteins were detected from the tubes in two distinct areas.Purification of glycoprotein using same condition of SDS-PAGE,same proteins were loaded,stained both with Coomassie Blue and with Gel Code Glycoprotein Staining Kit respectively. Under the condition,we successfully recovered glycoprotein bands from SDS-gel by passive diffusion of proteins. Glycoprotein bands recovered were detected by GelCodeGlycoprotein Staining Kit,and the result showed that recovery rate reached 50 percent,that the quantity of glycoprotein once purified from SDS-gel,was enough to be used in MS,HPLC,protein sequence analysis,glycan analysis,and characterization so on.In order to attain bioactive glycoprotein,glycoprotein of the leaves of Camellia Sinensis,was purified from coarse glycoproteins purified by Sephadex G-100 gel chromatography,by ConA-Sepharose 4B affinity chromatography. Purified glycoprotein was detected by GelCodeGlycoprotein Staining Kit,and the result indicated that it was a glycoprotein,because two glycoprotein bands appeared in gel,with their molecular weights 63000 and 54000 respectively.