Objective

1. Design of Genetic Sequences Encoding MMP-2-degradable Synthetic Recombinant Protein Kristen Kopf, RET Fellow 2010, Elk Grove High School Grace Cao, IMSA Fellow 2010 RET Mentor: Dr. Richard A. Gemeinhart NSF- RET Program Objective Abstract 1st Cycle 2nd Cycle Amplification 2c To develop cloning and PCR techniques for use in the creation of a gene encoding MMP-2-cleavable peptides. This gene will later be used in the creation of a biogel that will allow for targeted drug delivery. Protein-engineered biomaterials have the potential for drug targeted therapy against highly invasive and common primary brain tumors, such as glioblastomamultiforme. The repeating units of target recombinant protein polymers contain a binding and cleavage site for metalloproteinases (MMPs), which are excreted by tumor cells for tissue remodeling and angiogenesis. Polymer protein contact with MMPs will result in the degradation of the protein and the release of chemotherapeutic agents contained within the scaffold. The polymer protein was formed via primer-extension PCR, ligated into a plasmid cloning vector, and transformed into E. coli. This method allows for the creation of polymer proteins consisting of varying monomer repeats. The pool of technologies utilized here represents a promising approach for the development of protein-engineered biomaterials tailored for specific medical applications. Introduction • Need better treatment for glioblastomamultiforme, a highly invasive and common primary brain tumor1 • Possible solution is to insert a protein gel scaffold composed of peptides cleavable by matrix metalloproteinase-2 (MMP-2), an enzyme overactivated by the tumor2,3 • Biogel would contain chemotherapeutic agents and be inserted into the brain3 • MMP-2 would degrade the scaffold and release the chemotherapy in a targeted release3 • Investigation focuses on creating the gene for this protein • Consists of repeating MMP-2 cleavable sequences • Need enough repeats to code for a protein that can gel Results and Discussion MMP2- Cleavable Biogel MMP-2 cleavable peptide Chemotherapeutic agent Tumor cells releasing MMP-2 KEY -Template & - Primers - Added nucleotides First Reaction 5’-A B-3’ 3’-B’A’-5’ 5’-A B A-3’ 3’-B’A’-5’ Methods and Materials Figure 1. PCR primer Figure 2. Amplification of extension products. second PCR reaction Creation of Monomeric Polypeptide • Double-stranded oligonucleotides (51 bp) representing restriction enzyme sites and the partial DNA sequence of each strand of the target polypeptide monomers were designed and chemically synthesized by Integrated DNA Technologies, Inc. • Monomer DNA Sequence: • GGT CCG CTG GGC GTT CGT GGT • Monomer Amino Acid Sequence: • Glycine – Proline – Leucine – Glycine – Valine – Arginine – Glycine (GPLGVRG) Primer-Extension PCR • Purpose: To elongate the MMP-2-cleavable peptide sequence • Steps: • Ligation into pUC19c Vector • Restriction enzyme digest with SmaI • Ligate to vector with T4 DNA ligase in presence of SmaI • Transform E. coli with ligation product • Gel Electrophoresis of Insert • PCR amplification of insert using designed primers • Run product on agarose gel 5’-A B A -3’ 3’-A’B’A’-5’ 5’-A B-3’ 3’-A’B’A’ -5’ The product shown in Figure 1 from the second reaction was run on a 10% acrylamide gel, alongside a control sample that only contained the primers used for the reaction. Bands at roughly 25 and 50 base pairs (marked by blue arrows) can be seen in the lane containing the second PCR product. They represent the 1x insert, which is 21 base pairs in length, and the 2x insert, which is 42 base pairs long. The smear visible in the primers only lane resulted from primers annealing to each other, creating inserts of varying lengths. In Figure 2, the second reaction was further amplified. Second Reaction 5’-A BA -3’ 3’-A’B’-5’ 5’-A B A B-3’ 3’-A’B’-5’ 5’-A BA -3’ 3’-A’B’A’B’-5’ 5’-A BA B -3’ 3’-A’B’A’B’-5’ This is used as the template for the next reaction. Figure 3. PCR amplification of ligation into pUC19c vector. Products from the amplified second PCR primer extension reaction were ligated into pUC19c, the region surrounding the insert was amplified, and the resulting products were run on a 2.5% agarose gel. The area of amplification, without any inserts, was 220 base pairs long. Bands boxed in blue, which ran at about 300-350 base pairs long, represent 3-6 repeats of the MMP-2 cleavable peptide insert. • “A” = GGT CCG CTG, encoding GPL “B” = GGC GTT CGT GGT, encoding GVRG • Desired products are AB repeats • Only sense strand is shown, same process occurs with other strand Conclusion • PCR can be used to quickly generate genes of varying lengths. • This is a powerful tool that can later be used to generate protein polymers of varying sizes, in hopes that a certain length will be optimal for gel formation. References Acknowledgements 1. Uddin, S., & Jarmi, T. (2010). “Glioblastomamultiforme.” Retrieved 7/30/09, 2009. 2. Dai, B., Kang, S. H., Gong, W., Liu, M., Aldape, K. D., Sawaya, R., & Huang, S. (2007). Aberrant FoxM1B expression increases matrix metalloproteinase-2 transcription and enhances the invasion of glioma cells. Oncogene 6(42): 6212 - 6219. 3. Tauro, J. R., & Gemeinhart, R. A. (2005). Matrix metalloprotease triggered local delivery of cancer chemotherapeutics from hydrogel matrixes. Bioconjugate Chemistry16(5): 1133-1139. doi:10.1021/bc0501303 The IMSA and RET research was made possible by RET 2010 Program NSF Grant #CBET EEC-0743068, NIH R01 NS055095, and R03 EY014357 (RAG). This investigation was conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06 RR15482 from the National Center for Research Resources, NIH. We would also like to thank: Dr. Andreas Linninger, the RET Program Director; Dr. Richard Gemeinhart, Faculty Research Mentor; Jason Buhrman and Mary Tang, Graduate Research Mentors; and the University of Illinois at Chicago.