High Yield Expression of Recombinant CD151 in E. coli and a Structural Insight into Cholesterol Binding Domain
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.
KeywordsTransmembrane 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.
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.
- 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. https://doi.org/10.1074/jbc.M104041200.CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1091/mbc.01-10-0481.CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1091/mbc.01.CrossRefGoogle Scholar
- 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. https://doi.org/10.1016/j.bpj.2014.05.028.CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1002/art.22140.CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1016/j.yexcr.2008.04.011.CrossRefGoogle Scholar
- 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. https://doi.org/10.2353/ajpath.2008.071149.CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1016/j.rasd.2014.08.015.social.CrossRefGoogle Scholar
- 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. https://doi.org/10.1016/j.virol.2012.03.002.CrossRefPubMedGoogle Scholar
- 32.Palmer, J. S. (2015). Recombinant expression and analysis of tetraspanin extracellular-2 domains. Doctoral dissertation, University of Sheffield.Google Scholar
- 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. https://doi.org/10.1016/j.pep.2011.06.001.CrossRefPubMedPubMedCentralGoogle Scholar
- 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
- 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
- 39.Schrödinger. (2017). Schrödinger Release 2017-4: Prime, Schrödinger. New York: LLC.Google Scholar
- 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. https://doi.org/10.1371/journal.pone.0136507.CrossRefGoogle Scholar
- 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. https://doi.org/10.1074/jbc.m109.089078.CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1128/JVI.00559-12.CrossRefPubMedPubMedCentralGoogle Scholar