Purification of Tropomyosin, Paramyosin, Actin, Tubulin, Troponin and Kinases for Chemiproteomics and Its Application to Different Scientific Fields 英文参考文献.docVIP

PurificationofTropomyosin,Paramyosin,Actin,Tubulin, TroponinandKinasesforChemiproteomicsandIts ApplicationtoDifferentScientificFields TomasErban* LaboratoryofProteomics,DepartmentofStoredProductPestControlandFoodSafety,CropResearchInstitute,Prague,Czechia Abstract Background: p-aminobenzamidine (p-ABA) is used as a ligand in the purification of many serine proteases and in their removal from heterogeneous samples. Moreover, p-ABA has a potent ability to bind Ca -binding proteins. The binding abilityanduseofp-ABAinpurificationprocessesisstillnotfullyunderstood. 2+ Methodology/PrincipalFindings:Ap-Aminobenzamidine (p-ABA) ligand enabled thepurification ofthepanallergenic proteins tropomyosin and paramyosin, as well as actin, tubulin, troponin and several kinases and annexins, with variable specificity depending on the tissue source and slight modifications to the purification process. The high affinity of p-ABA to tropomyosin, paramyosin, actin, troponin and myosin is calcium-dependent, since calcium regulates the function ofthese proteins. Inaddition, p-ABA probably simulates phosphorylated serine and therefore purified appropriate kinases. Because p-ABA binds tocalcium-dependent proteins, and probably those with binding sites containing serine, it is not a suitable inhibitor of proteolysis during the purification of such proteins. p-ABA is widely used to inhibit proteases during protein purification processes, but it is used in columns here to purify non- protease proteins. Two strategies were applied; the first was the inactivation of proteases that were not of interest using protease inhibitors. The second strategy employed was the use of a Ca2+ wash solution to remove calcium- dependent proteins. The removal of calcium-dependent proteins from rabbit hind muscle pointed out even more selective purification. It is possible to obtain two purified samples: a) calcium dependent proteins and b) calcium independent proteins. Moreover, p-ABA may beuseful asamodel tos