Production of Recombinant Mouse Flower Protein in E. coli: Application of Mistic Fusion to Improving the Expression of Membrane Proteins

The structural and functional studies of membrane proteins have been greatly hindered due to difficulties in their over-expression and production. It is also often difficult to generate effective antibodies to membrane proteins. In our laboratory, we have addressed the problem of producing high level amounts of membrane proteins in Escherichia coli by use of Mistic, a Bacillus subtilis protein, as a fusion partner. Flower (Fwe) is a membrane protein that is conserved in animals and proposed to be a Ca2+ channel in neurons. It has been recently reported that in Drosophila Flower is a component of the cell competition response that is required and sufficient to label cells as “winners” or “losers”, promoting the elimination of weaker cells from a growing population in order to optimize tissue fitness. This process may have biomedical implications because imbalances in cell fitness appear during aging, cancer formation and metastasis. In this work, we employed the membrane protein Mistic to assist in the production of this protein. The construct 6xHis-Mistic – Flower carrying a TEV cleavage site was efficiently over expressed in E. coli. The IMAC-based purification and cleavage of the recombinant protein was achieved in the presence of the detergent lauryldimethylamino oxide (LDAO). Circular dichroism showed a high content in helical structure as predicted from the amino acid sequence by the program TMHMM. Also, the recombinant Mistic–Flower was used as antigen source to produce monoclonal antibodies in KO mice. The generated monoclonal antibodies were able to recognize Flower protein by Western blot and immunohistochemistry, indicating that this recombinant protein retained the antigenicity of the native form. These antibodies will facilitate importantly further functional studies on Flower protein.