Browsing by Author "Burgenson, David"
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Item Development of a Cell-Free Protein Expression System Derived from Human Blood Cells(2020-01-20) Burgenson, David; Rao, Govind; Chemical, Biochemical & Environmental Engineering; Engineering, Chemical and BiochemicalRecombinant proteins are typically produced using living cells. Alternatively, recombinant proteins can be produced using cellular extracts in a "cell-free" protein expression system. Cell-free protein expression systems are agile, robust, and some systems can produce mg/mL titers of target proteins within a matter of hours as opposed to days to weeks using traditional methods; supporting applications such as on-demand manufacturing of therapeutics at the point of care. Some of the first cell-free protein expression systems were derived from primary cells, specifically human lymphocytes and rabbit reticulocytes. However, with the improvement of yield in cell-free protein expression systems derived from cell lines, interest in cell-free protein expression systems derived from primary cells dropped. With the advancement of fields utilizing primary cells such as cell and gene therapy and the general improvement in yield from cell-free protein expression systems there is a renewed interest to develop a cell-free protein expression system derived from human blood cells. This dissertations outlines the development of a cell-free protein expression system derived from human blood cells, specifically peripheral blood mononuclear cells (PBMCs). Typically cell-free protein expression systems are derived from cells harvested in mid-log growth phase as this is where the cells are most translationally active. PBMCs are natively quiescent with a low rate of translation but can be stimulated to upregulate translation by cell activation. In the studies shown here, PBMCs are activated using the mitogenic substance PHA-M to produce a robust system capable of producing multiple recombinant proteins. In addition, the system is surveyed for improvement of recombinant protein expression by supplementing various small molecules and proteins shown to improve translation in similar cell-free protein expression systems. Using the methods developed in this dissertations, a maximum yield of 10 ng/mL of recombinant granulocyte colony stimulating factor (GCSF) was obtained using this cell-free protein expression system. While the yield of this system is currently insufficient to support the intended application of on demand manufacturing, these studies provide a proof of concept and baseline to support further studies to improve yield. Additionally, there are multiple basic science and research applications which may benefit from this technology.Item Rapid recombinant protein expression in cell-free extracts from human blood(Nature, 2018-06-22) Burgenson, David; Gurramkonda, Chandrasekhar; Pilli, Manohar; Ge, Xudong; Andar, Abhay; Kostov, Yordan; Tolosa, Leah; Rao, GovindSeveral groups have recently reported on the utility of cell-free expression systems to make therapeutic proteins, most of them employing CHO or E. coli cell-free extracts. Here, we propose an alternative that uses human blood derived leukocyte cell extracts for the expression of recombinant proteins. We demonstrate expression of nano luciferase (Nluc), Granulocyte-colony stimulating factor (G-CSF) and Erythropoietin (EPO) in cell-free leukocyte extracts within two hours. Human blood is readily available from donors and blood banks and leukocyte rich fractions are easy to obtain. The method described here demonstrates the ability to rapidly express recombinant proteins from human cell extracts that could provide the research community with a facile technology to make their target protein. Eventually, we envision that any recombinant protein can be produced from patient-supplied leukocytes, which can then be injected back into the patient. This approach could lead to an alternative model for personalized medicines and vaccines.