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 α-helix structures including a 3 kDa transmembrane domain.
Similar content being viewed by others
Abbreviations
- SFKs:
-
SRC family tyrosine kinases
- PAG:
-
phosphoprotein associated with glycosphingolipid-enriched microdomains
- CSK:
-
C-SRC tyrosine kinase
- GRP78:
-
78 kDa glucose-regulated protein
- ORP150:
-
150 kDa oxygen-regulated protein
References
Brdicka T., Pavlistova D., Leo A., Bruyns E., Korinek V., Angelisova P., Scherer J., Shevchenko A., Hilgert I., Cerny J., Drbal K., Kuramitsu Y., Kornacker B., Horejsi V., Schraven B. (2000). J. Exp. Med. 191: 1591–1604
Brdickova N., Brdicka T., Andera L., Spicka J., Angelisova P., Milgram S.L., Horejsi V. (2001). FEBS Lett. 507: 133–136
Carlino A., Toledo H., Vidal V., Redfield B., Strassman J., Abdel-Ghany M., Racker E., Weissbach H., Brot N. (1994). Biochem. Biophys. Res. Commun. 201: 1548–1553
Geourjon C., Deleage G. (1994). Protein Eng. 7: 157–164
Itoh K., Sakakibara M., Yamasaki S., Takeuchi A., Arase H., Miyazaki M., Nakajima N., Okada M., Saito T. (2002). J. Immunol. 168: 541–544
Kawabuchi M, Satomi Y., Takao T., Shimonishi Y., Nada S., Nagai K., Tarakhovsky A., Okada M. (2000). Nature. 404: 999–1003
Kuwabara K., Matsumoto M., Ikeda J., Hori O., Ogawa S., Maeda Y., Kitagawa K., Imuta N., Kinoshita T., Stern D. M., Yanagi H., Kamada T. (1996). J. Biol. Chem. 271: 5025–5032
Kyte J., Doolittle R. F. (1982). A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157: 105–132
Miyazaki S., Sugawara H., Ikeo K., Gojobori T., Tateno Y. (2004). Nucleic Acids Res. 32(Database issue): D31–D34
Takeuchi S., Takayama Y., Ogawa A., Tamura K., Okada M. (2000). J. Biol. Chem. 275: 29183–29186
Thompson J. D., Higgins D. G., Gibson T. J. (1994). Nucleic Acids Res. 22: 4673–4680
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Takeuchi, S. Expression and Purification of Human PAG, a Transmembrane Adapter Protein Using an Insect Cell Expression System and its Structure Basis. Protein J 25, 295–299 (2006). https://doi.org/10.1007/s10930-006-9015-6
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10930-006-9015-6