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Metalloprotease's Written Proposal

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  • 1. Sequence determination and Bioinformatics Analyses of Metalloproteases Domains cloned from Pongo pygmaeus Primate Genomic DNA Ana Velazquez and Dr. Michael Rubin RISE Program University of Puerto Rico at Cayey Department of Biology Abstract Metalloproteases are a group of proteases collectively responsible for the degradation of the extracellular matrix. They are important for the shape and modifications needed in the extracellular matrix. Metalloproteases are important in many aspects of biology, ranging from cell proliferation, differentiation and remodeling of the extracellular matrix (ECM) to vascularization and cell migration. Pongo pygmaeus is an orangutan specie native from Borneo. The genomic DNA of this specie is used for this experiment. The first objective in this investigation is to transform plasmid with MP gene to E. coli and grow these transformed cells with plasmid DNA, to purify the plasmid DNA from the culture, and perform plasmid miniprep. The long term goals are to send purified gene to sequence, determine the sequence of the MP gene in Pongo pygmaeus genomic DNA, and do a protease detection. The different methods used in this experiment are transformation, plasmid purification and plasmid miniprep preparation. Background differentiation and remodeling of the Metalloproteases are a group of proteases extracellular matrix (ECM) to collectively responsible for the degradation vascularization and cell migration. These of the extracellular matrix. They need a events occur several times during metal ion on their catalytic site for organogenesis in both normal development functioning. They are important for the and during tumor progression. These shape and modifications needed in the proteins are frequently studied, aimed extracellular matrix. Metalloproteases are specifically to study cell interaction and important in many aspects of biology, tumor formations. They are naturally ranging from cell proliferation, regulated by endogenous inhibitors. An
  • 2. imbalance between the active enzymes and more information about these frequently the natural inhibitors leads to the accelerated studied enzymes. destruction of connective tissue associated with the pathology of diseases such as Specific Aims arthritis, cancer, multiple sclerosis, and The first objective in this investigation is to cardiovascular diseases. transform plasmid with MP gene to E. coli Pongo pygmaeus is an orangutan specie and grow these transformed cells with native from Borneo. They live in the tree plasmid DNA in liquid culture for the tops and feed from fruits, bark, bird eggs, replication of the plasmid with the cells. and insects. The genomic DNA of this After the replication of the bacteria with the specie is used for this experiment because it desired plasmid is finished, the next step is is similar to human DNA and the results will to purify the plasmid DNA from the culture. be more accurate to the results it would have This will remove the plasmid out of the with human beings. By studying the MMP bacteria and discard the bacteria residues. gene in Pongo pygmaeus genomic DNA we The next desired aim after the purification of can control the irregularities in the MMP the plasmid is to perform plasmid minipreps, production. which is to produce small quantities of the Problem purified plasmid. In a long term aim the How can we control irregularities in project is going to be focused on sending the metalloprotease’s enzymatic activity? purified desired gene to bioinformatics and eventually do a protease detection. Hypothesis Short term goals Obtain a purified plasmid By the inhibition of the metalloprotease with desired gene to send for sequencing. gene, the irregularities of metalloprotease Long term goals Send purified gene to enzymes can be controlled. sequence, determine the sequence of the MP gene in Pongo pygmaeus genomic DNA, Significance and do a protease detection. This work is important because it would bring different options to health treatments for diseases affecting worldwide. It would also provide the scientific community with
  • 3. Procedures protocol used was provided by QIAprep® Miniprep Handbook. Transformation: Preliminary Studies This procedure will be done to insert the previously ligated plasmid with the desired The total of transformed bacteria colonies gene into the vector used, which is going to incubated in the agar plates at 37°C were 64 be E. coli. Then these same cells are going colonies in the plate containing 50µl of SOC to be cultivated in agar plates, replicating medium with bacteria and 540 colonies in with them the vector used. The protocol the plate containing 900µl of SOC medium followed for this procedure was provided by with bacteria. pGEM®-T and pGEM®-T Easy Vector References Systems Technical Manual. Clancy, J.P., Kong, M.Y.F., Gaggar, A., Plasmid Purification: Winkler, Y.L.M., Blalock, J.E. 2008. Matrix After the competent E. coli cells are Metalloproteinase Activity in Pediatric transformed and replicated, the replicated Acute Lung Injury. Med Sci. 2009; 6(1): 9– plasmid is purified from the whole cell. By 17. doing this it will provide only the plasmid Lengyel, E., Kenny, H.A., Kaur, S., with the metalloprotease gene. Coussens, L.M. 2008. The initial steps of ovarian cancer cell metastasis are mediated Plasmid Miniprep Preparation: by MMP-2 cleavage of vitronectin and The objective of this procedure is to produce fibronectin. Clin Invest. 2008 April 1; small quantities of the purified plasmid. This 118(4): 1367–1379. samples are the ones used in the Rubin, M.R., Quiñones, A.M., Electrophoresis gel analysis after going Rentas Torres, J. 2006. Biochemical through enzyme digestion. Other miniprep Detection, pharmacological inhibition, and samples are going to be the ones send for phylogenetic analysis of Caenorhabditis sequencing. The protocol followed for this elegans metalloproteases. Bios 77(4) procedure was the Quantum Prep™ Plasmid 113-126, 2006 Miniprep Kit Instruction Manual. For the Young, D.A., Milner, J.M., Rowan, samples that are going to be sequenced the A.D., Cawston, T.E. 2004.
  • 4. Metalloproteinase and inhibitor expression profiling of resorbing cartilage reveals pro-collagenase activation as a critical step for collagenolysis. Clin Invest. 2008 April 1; 118(4): 1367–1379

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