Tissue plasminogen activator (abbreviated tPA or PLAT) is a protein involved in the breakdown of blood clots. It is a serine protease (EC 3.4.21.68) found in endothelial cells that can be secreted into the plasma as well as the cells that line the blood vessels. As an enzyme, it catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for clot breakdown. Due to its ability of working on the clotting system, tPA is used in clinical medicine to treat embolic or thrombotic stroke. However, use is contraindicated in hemorrhagic stroke and head trauma.
We questioned the best possible was of measuring the tPA enzyme activity and sought the answer to this question by examining the yield of a tPA catalyzed reaction. We searched the sector and discovered the Human tPA Activity Kit of the company ASSAYPRO which we then ordered to use. In the reaction which catalyzes tPA, our aim was to show that tPA was active when plasminogen was transformed to plasmine.
To acquire the tPA gene, the tPA forward and tPA revers primers were synthesized from the cDNA’s we were in possession of. Using these primers, we acquired the tPA genes by performing the PCR of the cDNA. The head and neck cancer cell line was used as the source for cDNA.
GENE SYNTHESIS FROM cDNA OF 64A CELL LINE
Through the primers that we ordered and the cDNA, we acquired tPA inserts. The insert was, as expected, portraying that the base length was around 1700 bp.
Cloning Control 1
The specified primers were put into colony PCR. As it can be seen in the results above, a right insert was not achieved. This process was repeated several times but no result was achieved. Seeing that ligation did not provide a solution to the problem, synthetically produced inserts were ordered.
The tPA gene which was ligated with the synthetically synthesized Puc57 vector was cut using the EcorI and BamHI restriction enzymes and then ligated with pTRE vector to be transformed into the DH5-α strain. We again used Colony PCR to control the accuracy of our ligation.
CLONING CONTROL-2
Colony PCR was applied to the inserts acquired from transformation and ligation. As it can be seen in the figure above, the second colony contains the appropriate base length in respect to the ladder.
WESTERN BLOTTING
The bands in the Western Blot were accurate. The transfection of the inserts were verified and assays of tPA were prepared from the lysates.
After having proven the presence of tPA expression in the cells transfected with Western Blotting, the concentration of the amount of tPA in the cell and the amount of secreted tPA were measured using the ‘Human tPA Activity Kit’ to show that the expressed proteins are functionally active.
The real parameter of the measurement in the Assay was the product of the reaction of the plasmin enzyme. Since tPA shifts the inactive plasminogen to active plasmine, the measured value also presents tPA activity. The yield gives absorbance at 405 nm.
tPA ASSAY
According to the tPA assay results we obtained, we were able to prove that the tPA enzyme could be produced and secreted from the cell successfully hence also showing that tPA is functionally active.
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