Mutation of tandem repeats in fibrin's alpha-C region

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Sarah Elizabeth Spangler (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Cardiovascular problems such as myocardial infarctions and emboli are the number one killers in the United States. These health concerns arise from blood clotting unnecessarily in the veins or arteries. Studies have shown a correlation between these diseases and the mechanical properties of blood clots. Blood clots are strong and elastic because of fibrin , a protein found to be central to the formation and structure of blood clots. Our research focusses on determining the molecular origins of fibrin's elastic properties. We hypothesize that the C region of the  chain of fibrin (one of three chains;  ,  , and ) , which is unstructured , is the source of these characteristics. To test this hypothesis , we have used protein engineering to generate recombinant fibrinogen molecules. By mutating the C region in four different ways and comparing the new proteins to the original wild-type fibrin , we will be able to definitively prove whether the C region is responsible for fibrin's elasticity and which section of the C region holds that key sequence. Successfully producing and purifying a mutated fibrin protein with altered elasticity would provide a new and vital tool in further blood clot research. We also worked to determine the most productive and cost-efficient means of large-scale recombinant fibrinogen expression. Part one of this search focused on finding the most successful and least time-consuming means of protein production within cells. The most popular cell for mammalian protein expression is the Chinese Hamster Ovary (CHO) cell , but the process of expressing protein using CHO cells can take months. We compared the efficiency of CHO cells to HEKexpi293F cells. Next , we ran a series of tests to compare two transfection reagents (polyethylenimine (PEI) and expifectamine) to determine the most cost-effective reagent for the same amount of yield. Here we show that we can successfully express recombinant fibrinogen protein with ?-chain truncations , suggesting this is a viable approach to determining the origins of fibrin elasticity. The HEKexpi293F cells proved to be far more time- and cost-efficient than the CHO cells in producing recombinant fibrinogen. An altered protocol using PEI was found to be competitive with the standard expifectamine protocol for protein expression. The exact parameters for the protein purification process are still to be determined.

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Thesis

Language: English

Date: 2018

Keywords

Fibrin Blood Elastic

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