Development of a Cell-Free Protein Expression System Derived from Human Blood Cells

Abstract

Recombinant 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.