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.
Takeuchi
Satoru
S
Department of Protein Research, ProstaColon, 85 NE, Takamatsu, Kahoku, Ishikawa, 929-1215, Japan. prostacolon@yahoo.co.jp
D048868:Adaptor Proteins, Signal Transducing / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D007313:Insecta / Q000166:cytology; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data
D048868:Adaptor Proteins, Signal Transducing; D008565:Membrane Proteins; C498710:PAG1 protein, human