As our team objective is to provide a specific and efficient drug for colon cancer, 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 is able to produce and secrete anti-tumor molecule. Release of anti-tumor molecule to extracellular system is attained 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” of 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 BMP2 for 48 hours show significant decrease in cell growth. Hence, BMP2 is a potential drug to fight 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 delivery of BMP2 to the environment. To mimic the secretory activity of B. subtilis, we add signaling peptide type I gene, which works mostly through secretory pathway, at the upstream of Bmp2 gene in our construct (Tjalsma et al, 2000). That way, signaling peptide is 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 signal peptide is degraded by signal peptide peptidase (SPPases) (Tjalsma et al, 2000).

However, among hundreds of signaling peptide, we need to choose signaling peptide that allows the secretion of correct mature BMP2 in appropriate amount. Therefore, we choose YbdN, which is the highest efficiency signaling peptide and YdjM, the signaling peptide which support accurate cleavage from Spase. We had not been able to choose between the two choices until we construct two constructs consisting of YdjM or YbdN gene at the upstream of BMP2 gene and characterize both signaling peptide.

Although research has shown E. coli can produce BMP2 (Yuvaraj et al, 2012), no one has made a recombinant protein BMP2 in B. subtilis. Other than producing the recombinant protein, we need to 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.