Transcript Design of Genetic Sequences Encoding MMP-2-degradable

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
Protein-engineered biomaterials have the potential for drug targeted therapy
against highly invasive and common primary brain tumors, such as
glioblastoma multiforme. 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
•Need better treatment for glioblastoma multiforme, a highly invasive and
contained within the scaffold. The polymer protein was formed via primercommon primary brain tumor1
extension PCR, ligated into a plasmid cloning vector, and transformed into E.
•Possible solution is to insert a protein gel scaffold composed of peptides
cleavable by matrix metalloproteinase-2 (MMP-2), an enzyme overactivated by coli. This method allows for the creation of polymer proteins consisting of
varying monomer repeats. The pool of technologies utilized here represents
the tumor2,3
•Biogel would contain chemotherapeutic agents and be inserted into the brain3 a promising approach for the development of protein-engineered
biomaterials tailored for specific medical applications.
•MMP-2 would degrade the scaffold and release the chemotherapy in a
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.
Introduction
targeted release3
Results and Discussion
MMP2- Cleavable Biogel
MMP-2 cleavable peptide
Chemotherapeutic agent
Tumor cells releasing MMP-2
•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
Methods and Materials
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:
First Reaction
5’-A B-3’
3’-B’A’-5’
5’-A B A-3’
3’-B’A’-5’
5’-A B-3’
3’-A’B’A’ -5’
5’-A B A -3’ This is used as the template for
3’-A’B’A’-5’ the next reaction.
Second Reaction
5’-A B A -3’
3’-A’B’-5’
Figure 1. PCR primer
extension products.
Figure 2. Amplification of
second PCR reaction
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.
KEY
5’-A B A B-3’
3’-A’B’-5’
-Template
&
- Primers
- Added nucleotides
5’-A B A -3’
5’-A B A B -3’
3’-A’B’A’B’-5’
3’-A’B’A’B’-5’
•“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
Ligation into pUC19c Vector
1. Restriction enzyme digest with SmaI
2. Ligate to vector with T4 DNA ligase in presence of SmaI
3. Transform E. coli with ligation product
Gel Electrophoresis of Insert
1. PCR amplification of insert using designed primers
2. Run product on agarose gel
References
1. Uddin, S., & Jarmi, T. (2010). “Glioblastoma multiforme.” 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 Chemistry 16(5): 11331139. doi:10.1021/bc0501303
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.
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.
Acknowledgements
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.