As our team objective is to provide specific and efficient drug delivery against colon cancer, investigating and designing the way of drug synthesis and release is a significant part of our whole project. Hence this module is focusing on the production and delivery of anti-tumor drug. Synthesis of the drug is achieved by engineering bacteria to produce and secrete it. Release of the drug to the extracellular environment is performed by secretion of the recombinant gene product facilitated by attachment of a signaling peptide.

Among the hundreds of studied anti-tumor chemokines, BMP2 (bone morphogenetic protein 2) has caught our attention as the “drug” for our project. BMP2 is a signaling molecule in the BMP pathway, which belongs to the transforming growth factor beta superfamily. One function of the BMP pathway is to induce cell differentiation, especially in the development of bone and cartilage. BMP stimulates the formation of bone by inducing differentiation of bone cells. On the other hand, BMP2 has also been suggested to have high apoptotic activity towards colon cancer cells (Beck et al., 2005). According to Beck et al., colon cancer cells that are treated with 100 ng/mL BMP2 for 48 hours show significant decreases in cell growth. Knowing this fact, we see the potency of BMP2 as the drug to cure colon cancer. Therefore, we incorporate mature BMP2 gene in our construct and transform it into our chosen bacterial vector.

Furthermore, our choice of chassis is also important so we can ensure the secretion of BMP2. B. subtilis, which is a probiotic, was chosen as the chassis because of its harmless activity towards humans and its high secretory activity which is important for the delivery of BMP2 to the environment. To activate the secretory activity of our synthesized BMP2 in B. subtilis, we add signaling peptide type I gene - which works mostly through the B. subtilis' secretory pathway - upstream of the Bmp2 gene in our construct (Tjalsma et al., 2000). In this way, the signaling peptide would be translated together with BMP2 as a whole peptide, and be delivered to the cell membrane. Once it reaches the cell membrane, BMP2 is separated from the signaling peptide by signal peptidase (Spase). BMP2 is then transferred outside the cell and fold into its native conformation, while the signal peptide is degraded by signal peptide peptidase (SPPases) (Tjalsma et al., 2000)

However, among hundreds of signaling peptides, we need to choose the signaling peptide that allows the secretion of the accurate mature BMP2 polypeptide in appropriate amounts. Both YbdN, a signaling peptide of relatively high efficiency in B. subtilis, and YdjM, a peptide supporting reliably accurate cleavage from Spase, looked promising. It would be hard for us to make the decision just based on the literature, so we chose to build the two constructs consisting of ydjM or ybdN gene at the upstream of Bmp2 gene and compare them before making the final choice.

Although researchers have shown mature BMP2 can be produced in E. coli (Yuvaraj et al., 2012), recombinant BMP2 has not been successfully expressed in B. subtilis. Also, for the characterization of our work, other than checking whether we successfully produced the recombinant protein, we need to further confirm the function of BMP2 produced by our engineered bacteria.

Beck, S. E., Jung, B. H., Fiorino, A., Gomez, J., Del Rosario, E., Cabrera, B. L., Huang, S. C., Chow, J. Y. C., & Carethers J.M. (2006). Bone morphogenetic protein signaling and growth suppression in colon cancer. The American Journal of Physiology-Gastrointestinal and Liver Physiology, 291(1), G135-G145.

Ernesto, C. (2000). “Skeletal Growth Factors”. LIPPINCOTT WILLIAMS&WILKINS.

Hardwick,J.C., Van Den Brink,G.R., Bleuming,S.A., Ballester,I., Van Den Brande,J.M., Keller,J.J., Offerhaus,G.J., Van Deventer,S.J., Peppelenbosch,M.P., 2004.Bone morphogenetic protein 2 is expressed by, and acts upon, mature epithelial cells in the colon. Gastroenterology 2004. Jan. ;126. (1):111. -21. 126, 111-121.

Saravanan Yuvaraj, Sa’ad H. Al-Lahham, Rajesh Somasundaram, Patrick A. Figaroa, Maikel P. Peppelenbosch, and Nicolaas A. Bos, E. coli-Produced BMP-2 as a Chemopreventive Strategy for Colon Cancer: A Proof-of-Concept Study. Gastroenterology Research and Practice, vol. 2012, Article ID 895462, 6 pages, 2012. doi:10.1155/2012/895462

Tjalsma, H., Bolhuis, A., Jongbloed, J. D. H., Bron, S., & Dijl, J. M. V. (2000). Signal peptide-dependent protein transport in bacillus subtilis: a genome-based survey of the secretome. Microbiology and Molecular Biology Reviews, 64(3), 515-547.