Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Recombinant expression of a problematic protein (#122)

Larissa Doughty , Craig J Morton , Michael W Parker 1 2
  1. St. Vincent's Institute of Medical Research, Fitzroy, VIC, Australia
  2. Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia

CD151 is a member of the evolutionarily conserved tetraspanin family of transmembrane proteins, expressed in almost all cell types and tissues.[1, 2] The large extracellular loop (LEL) of CD151 is involved in protein-protein interactions that regulate cell adhesion, motility and proliferation in normal cells and has been found to participate in nearly all stages of cancer progression.[3-5] The LEL of CD151 exhibits high sequence variability from that of the other tetraspanins, making the LEL an attractive target for the development of interacting compounds that are selective for CD151 and affect its biological function. It has been shown that cell migration and metastasis is inhibited by anti-CD151 antibodies, strongly validating it as a target for therapeutic intervention.[6] 

In spite of the numerous in vivo studies of CD151, structural information of the LEL is limited to homology models. This is due to the usual difficulties associated with recombinant expression of eukaryotic membrane proteins in quantities suitable for structural studies. After trialling various expression strategies to produce recombinant CD151 LEL, with limited success, I have now developed a method to produce milligram quantities of purified monomeric protein suitable for crystallisation trials. This is a crucial step towards solving the structure of this therapeutically important protein.

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  2. Zoller, M., Tetraspanins: push and pull in suppressing and promoting metastasis. Nat Rev Cancer, 2009. 9(1): p. 40-55.
  3. Claas, C., et al., Association between the rat homologue of CO-029, a metastasis-associated tetraspanin molecule and consumption coagulopathy. J Cell Biol, 1998. 141(1): p. 267-80.
  4. Testa, J.E., et al., Eukaryotic expression cloning with an antimetastatic monoclonal antibody identifies a tetraspanin (PETA-3/CD151) as an effector of human tumor cell migration and metastasis. Cancer Res, 1999. 59(15): p. 3812-20.
  5. Kanetaka, K., et al., Overexpression of tetraspanin CO-029 in hepatocellular carcinoma. J Hepatol, 2001. 35(5): p. 637-42.
  6. Haeuw, J.F., et al., Tetraspanin CD151 as a target for antibody-based cancer immunotherapy. Biochem Soc Trans, 2011. 39(2): p. 553-8.