Production of O-GlcNAc Modified Recombinant Proteins in Escherichia coli

  • LIM, KI HONG (Graduate School of Biotechnology, Korea University) ;
  • CHANG HOON HA (Graduate School of Biotechnology, Korea University) ;
  • HYO IHL CHANG (Graduate School of Biotechnology, Korea University)
  • Published : 2002.04.01

Abstract

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslationally modified compound in eukaryotic cells. Human O-GlcNAc transferase (OGT) was produced as a maltose binding protein (MBP) fusion protein, which showed significant catalytic activity to modify recombinant Sp1, transcription factor. To facilitate the production of O-GlcNAc modified proteins, instead of using the tedious in vitro glycosylation reaction or expression in eukaryotic cells, a MBP-fusion OGT expression vector (pACYC184-MBPOGT) was constructed using pACYC184 plasmid, which could coexist with general prokaryotic expression vectors containing ColE1 origin. By cotransforming pACYC184-MBPOGT and pGEX-2T vectors into Escherichia coli BL21, intracellular O- GlcNAcylated proteins could be obtained by a simple purification procedure. It is expected that this may be a useful tool for production of O-GlcNAc modified proteins.

Keywords

References

  1. Akimoto, Y., L. K. Kreppel, H. Hirano, and G. W. Hart. 2000. Increased O-GlcNAc transferase in pancreas of rats with streptozotocin-induced diabetes. Diabetologia 43: 1239-1247
  2. Akimoto, Y., L. K. Kreppel, H. Hirano, and G. W. Hart. 1999. Localization of the O-linked N-acetylglucosamine transferase in rat pancreas. Diabetes 48: 2407-2413
  3. Akimoto, Y., L. K. Kreppel, H. Hirano, and G. W. Hart. 2001. Hyperglycemia and the O-GlcNAc transferase in rat aortic smooth muscle cells: Elevated expression and altered patterns of O-GlcNAcylation. Arch. Biochem. Biophys. 389: 166-175
  4. Bollag, D. M., M. D. Rozycki, and S. J. Edelstein. 1996. Protein Methods, 2nd ed., pp. 333-337. John Wiley and Sons, New York, U.S.A
  5. Chang, H.-A., J.-K. Chang, J.-W. Kim, and M.-N. Kim. 2000. Expression of heat shock protein 70 in umbilical vein endothelial cells infected by Staphylococcus aureus. J. Microbiol. Biotechnol. 10: 137-142
  6. Cheng, X., R. N. Cole, J. Zaia, and G. W. Hart. 2000. Alternative O-glycosylation/O-phosphorylation of the murine estrogen receptor beta. Biochemistry 39: 11609-11620
  7. Chou, T. Y., G. W. Hart, and C. V. Dang. 1995. c-Myc is glycosylated at threonine 58, a known phosphorylation site and a mutational hot spot in lymphomas. J. Biol. Chem. 270: 18961-18965
  8. Comer, F. I. and G. W. Hart. 2001. Reciprocity between OGlcNAc and O-phosphate on the carboxyl terminal domain of RNA polymerase II. Biochemistry 40: 7845-7852
  9. Gravel, P. and O. Golaz. 1996. In Walker, J. M. (ed.), The Protein Protocols Handbook, pp. 603-617. Humana Press, New Jersey, U.S.A
  10. Griffith, L. S., M. Mathes, and B. Schmitz. 1995. $\beta$ Amyloid precursor protein is modified with O-linked Nacetylglucosamine. J. Neurosci. Res. 41: 270-278.
  11. Guan, K. L. and J. E. Dixon. 1991. Eukaryotic proteins expressed in Escherichia coli: An improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biochem. 192: 262-267
  12. Haltiwanger, R. S., S. Busby, K. Grove, S. Li, D. Mason, L. Medina, D. Moloney, G. Philipsberg, and R. Scartozzi. 1997. O-Glycosylation of nuclear and cytoplasmic proteins: Regulation analogous to phosphorylation? Biochem. Biophys. Res. Commun. 231: 237-242
  13. Han, I., E.-S. Oh, and J. E. Kudlow. 2000. Responsiveness of the state of O-linked N-acetylglucosamine modification of nuclear pore protein p62 to the extracellular glucose concentration. Biochem. J. 350: 109-114
  14. Hanover, J. A. 2001. Glycan-dependent signaling: O-linked N-acetylglucosamine. FASEB J. 15: 1865-1876
  15. Huang, L., N. F. Mivechi, and D. Moskophidis. 2001. Insights into regulation and function of the major stressinduced hsp70 molecular chaperone in vivo: Analysis of mice with targeted gene disruption of the hsp70.1 or hsp70.3 gene. Mol. Cell. Biol. 21: 8575-8591
  16. Kearse, K. P. and G. W. Hart. 1991. Lymphocyte activation induces rapid changes in nuclear and cytoplasmic glycoproteins. Proc. Natl. Acad. Sci. USA 88: 1701-1705
  17. Kelly, W. G., M. E. Dahmus, and G. W. Hart. 1993. RNA polymerase II is a glycoprotein. Modification of the COOHterminal domain by O-GlcNAc. J. Biol. Chem. 268: 10416- 10424
  18. Konrad, R. J., I. Mikolaenko, J. F. Tolar, K. Liu, and J. E. Kudlow. 2001. The potential mechanism of the diabetogenic action of streptozotocin: Inhibition of pancreatic beta-cell O-GlcNAc-selective N-acetyl-b-D-glucosaminidase. Biochem. J. 356: 31-41
  19. Kreppel, L. K., M. A. Blomberg, and G. W. Hart. 1997. Dynamic glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats. J. Biol. Chem. 272: 9308-9315
  20. Lefebvre, T., C. Cieniewski, J. Lemoine, Y. Guerardel, Y. Leroy, Z.-P. Zanetta, and J.-C. Michalski. 2001. Identification of N-acetyl-D-glucosamine-specific lectins from rat liver cytosolic and nuclear compartments as heat-shock proteins. Biochem. J. 360: 179-188
  21. Lubas, W. A., D. W. Frank, M. Krause, and J. A. Hanover. 1997. O-Linked GlcNAc transferase is a conserved nucleocytoplasmic protein containing tetratricopeptide repeats. J. Biol. Chem. 272: 9316-9324
  22. Lubas, W. A. and J. A. Hanover. 2000. Functional expression of O-linked GlcNAc transferase. J. Biol. Chem. 275: 10983- 10988
  23. Okuno, Y., N. Imamoto, and Y. Yoneda. 1993. The transport of proteins into the nucleus requires the 70-kilodalton heat shock protein or its cytosolic cognate. Mol. Cell. Biol. 12: 2186-2192
  24. Reason, A. J., H. R. Morris, M. Panico, R. Marais, R. H. Treisman, R. S. Haltiwanger, G. W. Hart, W. G. Kelly, and A. Dell. 1992. Localization of O-GlcNAc modification on the serum response transcription factor. J. Biol. Chem. 267: 16911-16921
  25. Roos, M. D., K. Su, J. R. Baker, and J. E. Kudlow. 1997. OGlycosylation of an Sp1-derived peptide blocks known Sp1 protein interactions. Mol. Cell. Biol. 17: 6472-6480
  26. Ruther, A., N. Misawa, P. Boger, and G. Sandmann. 1997. Production of zeaxanthin in Escherichia coli transformed with different carotenogenic plasmids. Appl. Microbiol. Biotechnol. 48: 162-167
  27. Sayeski, P. P. and J. E. Kudlow. 1996. Glucose metabolism to glucosamine is necessary for glucose stimulation of transforming growth factor-alpha gene transcription. J. Biol. Chem. 271: 15237-15243
  28. Su, K., M. D. Roos, X. Yang, I. Han, A. J. Paterson, and J. E. Kudlow. 1999. An N-terminal region of Sp1 targets its proteasome-dependent degradation in vitro. J. Biol. Chem. 274: 15194-15202
  29. Yang, X., K. Su, M. D. Roos, Q. Chang, A. J. Paterson, and J. E. Kudlow. 2001. O-Linkage of N-acetylglucosamine to Sp1 activation domain inhibits its transcriptional capability. Proc. Natl. Acad. Sci. USA 98: 6611-6616
  30. Yao, P. J. and P. D. Coleman. 1998. Reduction of OLinked N-acetylglucosamine-modified assembly protein-3 in Alzheimer’s disease. J. Neurosci. 18: 2399-2411