As our team objective is to provide a specific and efficient drug delivery for 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 which are able to produce and secrete the anti-tumor chemicals. Releasing of the anti-tumor chemicals to extracellular environment is completed by secretion of recombinant gene product under the facilitation of signaling peptide.

Among hundreds of studied anti-tumor chemokine, BMP2, bone morphogenetic protein 2 has caught our attention as the “drug” for our project. BMP2 is a signaling molecule in BMP pathway, which belongs to the TGF-β superfamily. One function of BMP pathway is to induce cell differentiation, especially in the development of bone and cartilage. BMP stimulates the formation of bone by inducing the cell 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 which are treated with 100 ng/mL BMP-2 for 48 hours show significant decrease 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, choice of chassis also has important role to ensure the secretion of BMP2. B. subtilis, which is a probiotic, is chosen as the chassis because of its harmless activity towards human, and 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 B. subtilis' secretory pathway, at the upstream of Bmp2 gene in our construct (Tjalsma et al, 2000). In this way, signaling peptide would be translated together with BMP2 as a whole peptide, and delivered to the cell membrane. Once it reaches cell membrane, BMP2 is separated from 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 correct mature BMP2 in appropriate amounts. Therefore, we choose YbdN, which is the signaling peptide of the highest efficiency in B. subtilis and YdjM, the signaling peptide which supports accurate cleavage from Spase. It would be hard for us just 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 characterize both signaling peptide before making the final choice.

Although researchers have shown mature BMP2 can be produced in E. coli (Yuvaraj et al, 2012), no one has repoorted to make a recombinant protein BMP2 successfully 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.

References
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.