Biochemistry (Moscow)

, Volume 82, Issue 2, pp 186–191 | Cite as

High level soluble expression and ATPase characterization of human heat shock protein GRP78

  • Shuang Wu
  • Hongpeng Zhang
  • Miao Luo
  • Ke Chen
  • Wei Yang
  • Lei Bai
  • Ailong Huang
  • Deqiang WangEmail author


Human GRP78 has been shown to promote cancer progression and is regarded as a novel target for anticancer drugs. However, generation of recombinant full-length GRP78 remains challenging. This report demonstrates that E. coli autoinduction is an excellent method for the preparation of active recombinant GRP78 protein. The final yield was approximately 50 mg/liter of autoinduction culture. Gel-filtration experiments confirmed that the chaperone is a monomer. The purified human GRP78 catalyzed the conversion of ATP to ADP without requiring metal ions as cofactors. Three mutants, T38A, T229A, and S300A, exhibited much lower activity than wild-type GRP78, indicating that the active sites of the ATPase are located at the negatively charged cavity. Three mutants in the negatively charged cavity region dramatically reduced GRP78 activity, further confirming the region as the site of ATPase activity.


GRP78 ATPase autoinduction monomer active site 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

10541_2017_399_MOESM1_ESM.pdf (133 kb)
Supplementary material, approximately 133 KB.


  1. 1.
    Chevalier, M., Rhee, H., Elguindi, E. C., and Blond, S. Y. (2000) Interaction of murine BiP/GRP78 with the DnaJ homologue MTJ1, J. Biol. Chem., 275, 19620–19627.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Hendershot, L. M. (2004) of ERfunction, Mt. Sinai J. Med., 71, 289–297.PubMedGoogle Scholar
  3. 3.
    Roller, C., and Maddalo, D. (2013) The molecular chaperone GRP78/BiP in the development of chemoresistance: mechanism and possible treatment, Front. Pharmacol., 4, 10.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Gorbatyuk, M. S., and Gorbatyuk, O. S. (2013) The molecular chaperone GRP78/BiP as a therapeutic target for neurodegenerative disorders: a mini review, J. Genet. Syndr. Gene Ther., 4, 128.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Triantafilou, K., Fradelizi, D., Wilson, K., and Triantafilou, M. (2002) GRP78, a coreceptor for coxsackievirus A9, interacts with major histocompatibility complex class I molecules which mediate virus internalization, J. Virol., 76, 633–643.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Shin, B. K., Wang, H., Yim, A. M., Le Naour, F., Brichory, F., Jang, J. H., Zhao, R., Puravs, E., Tra, J., Michael, C. W., Misek, D. E., and Hanash, S. M. (2003) Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function, J. Biol. Chem., 278, 7607–7616.CrossRefPubMedGoogle Scholar
  7. 7.
    Luo, S., Mao, C., Lee, B., and Lee, A. S. (2006) GRP78/BiP is required for cell proliferation and protecting the inner cell mass from apoptosis during early mouse embryonic development, Mol. Cell. Biol., 26, 5688–5697.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Zhang, X. (2010) Roles of GRP78 in physiology and cancer, J. Cell. Biochem., 110, 1299–1305.CrossRefPubMedGoogle Scholar
  9. 9.
    Ni, M., Zhang, Y., and Lee, A. S. (2011) Beyond the endoplasmic reticulum: atypical GRP78 in cell viability, signaling and therapeutic targeting, Biochem. J., 434, 181–188.PubMedGoogle Scholar
  10. 10.
    Li, H., Song, H., Luo, J., Liang, J., Zhao, S., and Su, R. (2012) Knockdown of glucose-regulated protein 78 decreases 1 the invasion, metalloproteinase expression and ECM degradation in hepatocellular carcinoma cells, J. Exp. Clin. Cancer Res., 31, 39.PubMedGoogle Scholar
  11. 11.
    Su, R., Li, Z., Li, H., Song, H., Bao, C., Wei, J., and Cheng, L. (2010) GRP78 promotes the invasion of hepatocellular carcinoma, BMC Cancer, 10, 20.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Cook, K. L., Clarke, P. A., and Clarke, R. (2013) Targeting GRP78 and antiestrogen resistance in breast cancer, Future Med. Chem., 5, 1047–1057.CrossRefPubMedGoogle Scholar
  13. 13.
    Carlino, A., Toledo, H., Skaleris, D., DeLisio, R., Weissbach, H., and Brot, N. (1992) Interactions of liver Grp78 and Escherichia coli recombinant Grp78 with ATP: multiple species and disaggregation, Proc. Natl. Acad. Sci. USA, 89, 2081–2085.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kozutsumi, Y., Normington, K., Press, E., Slaughter, C., Sambrook, J., and Gething, M. J. (1989) Identification of immunoglobulin heavy chain binding protein as glucoseregulated protein 78 on the basis of amino acid sequence, immunological cross-reactivity, and functional activity, J. Cell. Sci., 11, 115–137.Google Scholar
  15. 15.
    Ciplys, E., Aucynaite, A., and Slibinskas, R. (2014) Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae, Microb. Cell. Fact., 13, 22.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Studier, F. W. (2005) Protein production by autoinduction in high density shaking cultures, Protein Expr. Purif., 41, 207–234.CrossRefPubMedGoogle Scholar
  17. 17.
    Studier, F. W. (2014) Stable expression clones and autoinduction for protein production in E. coli, Methods Mol. Biol., 1091, 17–32.CrossRefPubMedGoogle Scholar
  18. 18.
    Halford, M. M., He, Y. C., and Stacker, S. A. (2014) Expression and purification of bioactive, low-endotoxin recombinant human vitronectin, Biotechniques, 56, 331–333.PubMedGoogle Scholar
  19. 19.
    Li, J., Hua, Z., Miao, L., Jian, T., Wei, Y., Shasha, Z., Shaocheng, Z., Zhen, G., Hongpeng, Z., Ailong, H., and Deqiang, W. (2013) The crystal structure and biochemical properties of DHBPS from Streptococcus pneumoniae, a potential anti-infective target for 1 Gram-positive bacteria, Protein Expr. Purif., 91, 161–168.PubMedGoogle Scholar
  20. 20.
    Henkel, R. D., VandeBerg, J. L., and Walsh, R. A. (1988) A microassay for ATPase, Anal. Biochem., 169, 312–318.CrossRefPubMedGoogle Scholar
  21. 21.
    Lin, J., Eggensperger, S., Hank, S., Wycisk, A. I., Wieneke, R., Mayerhofer, P. U., and Tampe, R. (2014) A negative feedback modulator of antigen processing evolved from a frameshift in the cowpox virus genome, PLoS Pathog., 10, e1004554.Google Scholar
  22. 22.
    Birkholtz, L. M., Blatch, G., Coetzer, T. L., Hoppe, H. C., Human, E., Morris, E. J., Ngcete, Z., Oldfield, L., Roth, R., Shonhai, A., Stephens, L., and Louw, A. I. (2008) Heterologous expression of plasmodial proteins for structural studies and functional annotation, Malar. J., 7, 197.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Blond-Elguindi, S., Fourie, A. M., Sambrook, J. F., and Gething, M. J. (1993) Peptide-dependent stimulation of the ATPase activity of the molecular chaperone BiP is the result of conversion of oligomers to active monomers, J. Biol. Chem., 268, 12730–12735.PubMedGoogle Scholar
  24. 24.
    Freiden, P. J., Gaut, J. R., and Hendershot, L. M. (1992) Interconversion of three differentially modified and assembled forms of BiP, EMBO J., 11, 63–70.PubMedPubMedCentralGoogle Scholar
  25. 25.
    Wisniewska, M., Karlberg, T., Lehtio, L., Johansson, I., Kotenyova, T., Moche, M., and Schuler, H. (2010) Crystal structures of the ATPase domains of four human Hsp70 isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/ Hsp70B’, and HSPA5/BiP/GRP78, PLoS One, 5, e8625.CrossRefGoogle Scholar
  26. 26.
    Macias, A. T., Williamson, D. S., Allen, N., Borgognoni, J., Clay, A., Daniels, Z., Dokurno, P., Drysdale, M. J., Francis, G. L., Graam, C. J., Howes, R., Matassova, N., Murray, J. B., Parsons, R., Shaw, T., Surgenor, A. E., Terry, L., Wang, Y., Wood, M., and Massey, A. J. (2011) Adenosine-derived inhibitors of 78 kDa glucose regulated protein (Grp78) ATPase: insights into isoform selectivity, J. Med. Chem., 54, 4034–4041.CrossRefPubMedGoogle Scholar
  27. 27.
    DeLano, W. L. (2004) The PyMOL User’s Manual, Delano Scientific, San Carlos, CA.Google Scholar

Copyright information

© Pleiades Publishing, Ltd. 2017

Authors and Affiliations

  • Shuang Wu
    • 1
    • 2
  • Hongpeng Zhang
    • 1
    • 2
  • Miao Luo
    • 3
  • Ke Chen
    • 1
  • Wei Yang
    • 2
  • Lei Bai
    • 1
  • Ailong Huang
    • 1
  • Deqiang Wang
    • 1
    • 2
    Email author
  1. 1.Key Laboratory of Molecular Biology of Infectious DiseaseChongqing Medical UniversityChongqingPeople’s Republic of China
  2. 2.Department of Laboratory MedicineChongqing Medical UniversityChongqingPeople’s Republic of China
  3. 3.Department of Laboratory MedicinePeople’s Hospital of YuBei DistrictChongqingPeople’s Republic of China

Personalised recommendations