Molecular Biotechnology

, Volume 61, Issue 12, pp 905–915 | Cite as

High Yield Expression of Recombinant CD151 in E. coli and a Structural Insight into Cholesterol Binding Domain

  • Gayathri Purushothaman
  • Vijay ThiruvenkatamEmail author
Original paper


CD151 is an abundantly expressed eukaryotic transmembrane protein on the cell surface. It is involved in cell adhesion, angiogenesis and signal transduction as well in disease conditions such as cancer and viral infections. However, the molecular mechanism of CD151 activation is poorly understood due to the lack of structural information. By considering the difficulties in expressing the membrane protein in E. coli, herein we introduce the strategic design for the effective expression of recombinant CD151 protein in E. coli with high yield, that would aid for the structural studies. CD151 having four transmembrane domain (TMD’s) along with small and a large extracellular loop (LEL) is constructed in parts to enhance the soluble expression of the protein attached with fusion tag. This has led to the high yield of the recombinant CD151 protein in the designed constructs. The recombinant CD151 protein is characterized and confirmed by western blot, CD and Mass peptide fingerprint. The molecular dynamics simulations (MDS) for the full-length CD151 shows conformational changes in the LEL of the protein in the presence and absence of cholesterol and indicate the certainty of closed and open conformation of CD151 based on cholesterol binding. The MDS results have led to the understanding of the possible underlying mechanism for the activation of the CD151 protein.


Transmembrane protein CD151 Construct design Recombinant expression Molecular dynamics simulation Cholesterol binding domain 



The authors thank SERB Project No.: EMR/2016/001,022 for funding and IIT Gandhinagar for infrastructure. We also thank Dr Sivapriya Kirubakaran for her constant support and helpful discussions, and Althaf Shaik and Deekshi Angira for proofreading the manuscript.

Author Contributions

VT conceptualized the idea and designed the project. VT and GP designed all the experiments and protocols. GP performed all the experiments. VT and GP wrote the manuscript together.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Supplementary material

12033_2019_212_MOESM1_ESM.docx (6.4 mb)
Supplementary material 1 (DOCX 6528 kb)


  1. 1.
    Fitter, S., Tetaz, T. J., Berndt, M. C., & Ashman, L. K. (1995). Molecular cloning of cDNA encoding a novel platelet-endothelial cell tetra-span antigen, PETA-3. Blood, 86, 1348–1355.CrossRefGoogle Scholar
  2. 2.
    Hasegawa, H., Utsunomiya, Y., Kishimoto, K., Kohsuke, Y., & Fujita, S. (1996). SFA-1, a novel cellular gene induced by human T-cell leukemia virus type 1, is a member of the transmembrane 4 superfamily. Journal of Virology, 70, 3258–3263.PubMedPubMedCentralGoogle Scholar
  3. 3.
    Sincock, P. M., Mayrhofer, G., & Ashman, L. K. (1997). Localization of the transmembrane 4 superfamily (TM4SF) member PETA-3 (CD151) in normal human tissues: comparison with CD9, CD63, an integrin. The Journal of Histochemistry & Cytochemistry, 45, 515–525.CrossRefGoogle Scholar
  4. 4.
    Hemler, M. E. (2005). Tetraspanin functions and associated microdomains. Nature Reviews Molecular Cell Biology, 6, 801–811. Scholar
  5. 5.
    Chakraborty, D., Rodgers, K. K., Conley, S. M., & Naash, M. I. (2013). Structural characterization of the second intradiscal loop of the photoreceptor tetraspanin RDS. The FEBS Journal, 280, 127–138. Scholar
  6. 6.
    Lu, J., Li, J., Liu, S., Wang, T., Ianni, A., & Bober, E. (2017). Exosomal tetraspanins mediate cancer metastasis by altering host microenvironment. Oncotarget, 8, 62803–62815.PubMedPubMedCentralGoogle Scholar
  7. 7.
    Berditchevski, F., Gilbert, E., Griffiths, M. R., Fitter, S., Ashman, L., & Jenner, S. J. (2001). Analysis of the CD151 α3β1 integrin and CD151 tetraspanin interactions by mutagenesis. The Journal of Biological Chemistry, 276, 41165–41174. Scholar
  8. 8.
    Zhang, X. A., Kazarov, A. R., Yang, X., Bontrager, A. L., Stipp, C. S., & Hemler, M. E. (2002). Function of the tetraspanin CD151 α6β1 integrin complex during cellular morphogenesis. Molecular Biology of the Cell, 13, 1–11. Scholar
  9. 9.
    Yang, X., Claas, C., Kraeft, S., Chen, L. B., Wang, Z., Kreidberg, J. A., et al. (2002). Palmitoylation of tetraspanin proteins: modulation of CD151 lateral interactions. Subcellular Distribution, and Integrin-Dependent Cell Morphology, 13, 767–781. Scholar
  10. 10.
    Homsi, Y., Schloetel, J., Scheffer, K. D., Schmidt, T. H., Destainville, N., Florin, L., et al. (2014). The extracellular domain is essential for the formation of CD81 tetraspanin webs. Biophysical Journal, 107, 100–113. Scholar
  11. 11.
    Zimmerman, B., Kelly, B., Mcmillan, B. J., Seegar, T. C. M., Dror, R. O., Kruse, A. C., et al. (2016). Crystal structure of a full-length human tetraspanin reveals a cholesterol-binding pocket t. Cell, 167, 1041.e11–1045.e11. Scholar
  12. 12.
    Yáñez-Mó, M., Barreiro, O., Gordon-Alonso, M., Sala-Valdés, M., & Sánchez-Madrid, F. (2009). Tetraspanin-enriched microdomains: a functional unit in cell plasma membranes. Trends in Cell Biology, 19, 434–446. Scholar
  13. 13.
    Yauch, R. L., Kazarov, A. R., Desai, B., Lee, R. T., & Hemler, M. E. (2000). Direct extracellular contact between integrin α3β1 and TM4SF Protein CD151. The Journal of Biological Chemistry, 275, 9230–9238.CrossRefGoogle Scholar
  14. 14.
    Lammerding, J., Kazarov, A. R., Huang, H., Lee, R. T., & Hemler, M. E. (2003). Tetraspanin CD151 regulates α6β1 integrin adhesion strengthening. Proceedings of the National Academy of Sciences, 100, 7616–7621.CrossRefGoogle Scholar
  15. 15.
    Fujita, Y., Shiomi, T., Yanagimoto, S., Matsumoto, H., & Toyama, Y. (2006). Tetraspanin CD151 is expressed in osteoarthritic cartilage and is involved in pericellular activation of Pro—matrix metalloproteinase 7 in osteoarthritic chondrocytes. Arthritis and Rheumatism, 54, 3233–3243. Scholar
  16. 16.
    Sachs, N., Kreft, M., van der Bergh Weerman, M. A., Beynon, A. J., Peters, T. A., Weening, J. J., et al. (2006). Kidney failure in mice lacking the tetraspanin CD151. Journal of Cell Biology, 175, 33–39. Scholar
  17. 17.
    Cowin, A. J., Adams, D., Geary, S. M., Wright, M. D., Jones, J. C. R., & Ashman, L. K. (2006). Wound healing is defective in mice lacking tetraspanin CD151. The Journal of Investigative Dermatology, 126, 680–689. Scholar
  18. 18.
    Geary, S. M., Cowin, A. J., Copeland, B., Baleato, R. M., Miyazaki, K., & Ashman, L. K. (2008). The role of the tetraspanin CD151 in primary keratinocyte and fibroblast functions: Implications for wound healing. Experimental Cell Research, 4, 2165–2175. Scholar
  19. 19.
    Baleato, R. M., Guthrie, P. L., Gubler, M.-C., Ashman, L. K., & Roselli, S. (2008). Deletion of CD151 results in a strain-dependent glomerular disease due to severe alterations of the glomerular basement membrane. The American Journal of Pathology., 173, 927–937. Scholar
  20. 20.
    Baldwin, G., Novitskaya, V., Sadej, R., Pochec, E., Litynska, A., Hartmann, C., et al. (2008). Tetraspanin CD151 Regulates Glycosylation of α3β1 Integrin. The Journal of Biological Chemistry, 283, 35445–35454. Scholar
  21. 21.
    Yang, X. H., Richardson, A. L., Maria, P. Z., Torres-Arzayus, I., Sharma, C., Kazarov, A. R., et al. (2015). CD151 accelerates breast cancer by regulating α6 integrin function, signaling, and molecular organization. Cancer Research, 8, 1699–1712. Scholar
  22. 22.
    Li, Q., Yang, X. H., Xu, F., Sharma, C., Wang, H., Knoblich, K., et al. (2013). carcinomaTetraspanin CD151 plays a key role inskin squamous cell carcinoma. Oncogene, 32, 617–632. Scholar
  23. 23.
    Kwon, M. J., Seo, J., Kim, Y. J., Kwon, M. J., Choi, J. Y., Kim, T., et al. (2013). Lung cancer prognostic significance of CD151 overexpression in non-small cell lung cancer. Lung Cancer, 81, 109–116. Scholar
  24. 24.
    Tokuhara, T., Hasegawa, H., Hattori, N., Ishida, H., Taki, T., Tachibana, S., et al. (2001). Clinical significance of CD151 gene expression in non-small cell. Clinical Cancer Research, 7, 4109–4114.PubMedGoogle Scholar
  25. 25.
    Copeland, B. T., Bowman, M. J., & Ashman, L. K. (2013). Genetic ablation of the tetraspanin CD151 reduces spontaneous metastatic spread of prostate cancer in the TRAMP model. Molecular Cancer Research: MCR, 11, 95–106. Scholar
  26. 26.
    Deng, X., Li, Q., Hoff, J., Novak, M., Yang, H., Jin, H., et al. (2012). Integrin-associated CD151 drives ErbB2-evoked mammary tumor. Neoplasia, 14, 678–689. Scholar
  27. 27.
    Yue, S., Mu, W., & Zo, M. (2013). Tspan8 and CD151 promote metastasis by distinct mechanisms q. European Journal of Cancer, 49, 2934–2948. Scholar
  28. 28.
    Van Spriel, A. B., & Figdor, C. G. (2010). The role of tetraspanins in the pathogenesis of infectious diseases. Microbes and Infection, 12, 106–112. Scholar
  29. 29.
    Scheffer, K. D., Gawlitza, A., Spoden, G. A., Zhang, X. A., Lambert, C., Berditchevski, F., et al. (2013). Tetraspanin CD151 mediates papillomavirus type 16 endocytosis. Journal of Virology, 87, 3435–3446. Scholar
  30. 30.
    Zhu, Y., Luo, Y., Cao, M., Liu, Y., Liu, X., Wang, W., et al. (2012). Significance of palmitoylation of CD81 on its association with tetraspanin-enriched microdomains and mediating hepatitis C virus cell entry. Virology, 429, 112–123. Scholar
  31. 31.
    Kitadokoro, K., Bordo, D., Galli, G., Petracca, R., Falugi, F., Abrignani, S., et al. (2001). CD81 extracellular domain 3D structure: Insight into the tetraspanin superfamily structural motifs. The EMBO Journal, 20, 12–18.CrossRefGoogle Scholar
  32. 32.
    Palmer, J. S. (2015). Recombinant expression and analysis of tetraspanin extracellular-2 domains. Doctoral dissertation, University of Sheffield.Google Scholar
  33. 33.
    Wagner, S., Bader, M. L., Drew, D., & De Gier, J. (2006). Rationalizing membrane protein overexpression. Trends in Biotechnology, 24, 364–371. Scholar
  34. 34.
    Hu, J., Qin, H., Philip, F., & Cross, T. A. (2011). A systematic assessment of mature MBP in membrane protein production: Overexpression, membrane targeting and purification. Protein Expression and Purification, 80, 34–40. Scholar
  35. 35.
    Kitadokoro, K., Ponassi, M., Galli, G., & Bolognesi, M. (2002). Subunit association and conformational flexibility in the head subdomain of human CD81 large extracellular loop. Biological Chemistry, 389, 1447–1452.Google Scholar
  36. 36.
    Bujotzek, A., Lipsmeier, F., Harris, S. F., Benz, J., Georges, G., Bujotzek, A., et al. (2016). VH-VL orientation prediction for antibody humanization candidate selection: A case study. MAbs, 8, 288–305. Scholar
  37. 37.
    Jia, X., Schulte, L., Loukas, A., Pickering, D., Pearson, M., Mobli, M., et al. (2014). Solution structure, membrane interactions, and protein binding partners of the tetraspanin Sm-TSP-2, a vaccine antigen from the human blood fluke schistosoma. Journal of Biological Chemistry, 289, 7151–7163.CrossRefGoogle Scholar
  38. 38.
    Cunha, E. S., Sfriso, P., Rojas, A. L., Roversi, P., Hospital, A., & Orozco, M. (2017). Mechanism of structural tuning of the Hepatitis C virus human cellular receptor CD81 large extracellular loop. Structure., 25, 53–65. Scholar
  39. 39.
    Schrödinger. (2017). Schrödinger Release 2017-4: Prime, Schrödinger. New York: LLC.Google Scholar
  40. 40.
    Harder, E., Damm, W., Maple, J., Wu, C., Reboul, M., Xiang, J. Y., et al. (2016). OPLS3: A force field providing broad coverage of drug-like small molecules and proteins. Journal of Chemical Theory and Computation, 12, 281–296. Scholar
  41. 41.
    Jorgensen, W. L., Maxwell, D. S., & Tirado-rives, J. (1996). Development and testing of the OPLS All-atom force field on conformational energetics and properties of organic liquids. Journal of Americal Chemical Soceity., 7863, 11225–11236. Scholar
  42. 42.
    Wagner, S., Klepsch, M. M., Schlegel, S., Appel, A., Draheim, R., Tarry, M., et al. (2008). Tuning Escherichia coli for membrane protein overexpression. Proceedings of the National Academy of Sciences, 105, 14371–14376.CrossRefGoogle Scholar
  43. 43.
    Miroux, B., & Walker, J. E. (1996). Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. Journal of Molecular Biology, 260, 289–298.CrossRefGoogle Scholar
  44. 44.
    Gong, Z., Martin-garcia, J. M., & Daskalova, S. M. (2015). Biophysical characterization of a vaccine candidate against HIV-1: The transmembrane and membrane proximal domains of HIV-1 gp41 as a maltose binding protein fusion. PLoS ONE, 10(8), 1–22. Scholar
  45. 45.
    Gould, A. D., & Shilton, B. H. (2010). Studies of the maltose transport system reveal a mechanism for coupling ATP hydrolysis to substrate translocation without direct recognition of substrate. Journal of Biological Chemistry, 285, 11290–11296. Scholar
  46. 46.
    Rajesh, S., Sridhar, P., Tews, B. A., Feneant, L., Cocquerel, L., Ward, D. G., et al. (2012). Structural basis of ligand interactions of the large extracellular domain of tetraspanin CD81. Journal of Virology, 86, 9606–9616. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Discipline of Biological EngineeringIndian Institute of Technology GandhinagarGandhinagarIndia

Personalised recommendations