David Burgenson - PhD Proposal Defense

Title: Producing Recombinant Proteins Using a Cell Free Protein Expression System Derived From Human Blood

Candidate: David Burgenson

Date: 8/8/2019

Location: TRC 206

Time: 2 pm 

Cell free protein expression is a technique which has been used extensively to study protein expression and has helped scientists to understand the underlying mechanisms for decades. Recently, advancements in cell free protein synthesis platforms have supported manufacturing of therapeutic and vaccine proteins ranging from a portable single dose of an anti-radiation medication, to a thousand-liter reaction to produce oncology drugs. One of the early in-vitro translation cell free protein expression systems used rabbit reticulocytes (immature red blood cells) as a source of cells to produce active cell free extract. Using this system as a source of inspiration, our group began looking for cells present in human blood that could be used to produce active cell free extract. In a recent publication our group showed that leukocyte enriched blood products could be used to produce an active transcription and translation cell free extract that was able to produce a reporter protein (Nano-Luciferase, NLuc) and two therapeutic proteins (Granulocyte Colony Stimulating Factor, GCSF, and erythropoietin, EPO). However, the concentration of protein produced by this initial system was less than 10 ng/mL, far below the threshold necessary for therapeutic use. It is believed at least some of the cells responsible for providing protein expression machinery are peripheral lymphocytes, which are natively quiescent and have multiple molecular mechanisms for repressing protein expression. Reversing the quiescent state of these peripheral lymphocytes has been shown to produce a more active in-vitro translation system but has never been applied to coupled transcription and translation system. The goal of this project is to increase the yield of a cell free protein expression system derived from human blood, which could be used to produce therapeutic or vaccine molecules that could be administered autologously. As the “host cell proteins” of this systems are autologous, the chance for immunogenic response is minimized, and therefore purification burden could be minimal. To achieve this goal, we propose the following specific aims: 1) Isolate and activate cells from human blood that support cell free protein synthesis. 2) Produce a vaccine molecule using a cell free protein synthesis system derived from human blood. 3)  Investigate the effects of reactor and reaction configuration on yield in a cell free protein synthesis system derived from human blood.