Expression and Purification of Human PAG, a Transmembrane Adapter Protein Using an Insect Cell Expression System and its Structure Basis.
In this study, we report the purification and structure basis of human phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a C-SRC tyrosine kinase (CSK)-binding protein. Human PAG was produced using an insect cell expression system. The PAG was purified by metal affinity, ion exchange, and gel filtration chromatographies. The final purity of gel-purified PAG was evaluated by SDS-PAGE and mass spectrometry. Recombinant human PAG migrates to 60 kDa on SDS-PAGE gel, while native PAG is a 46 kDa transmembrane adapter protein in lipid rafts. Recombinant human PAG has a difference of 2590.7 Da with a calculated mass (47803.41 Da) and an observed mass (50394.1 Da) by mass spectrometry. Consequently, although human PAG sequence shares well-known sites for modifications such as myristoylation, palmitoylation, and tyrosine phosphorylation sites, perhaps the difference suggests the existence of unknown modification sites. We show the high PAG- binding ability with CSK in vitro as well as the human PAG structure characterized by 11 alpha-helix structures including a 3 kDa transmembrane domain.[1]References
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