Date(s) - 04/04/2013
Methods for rapidly expressing proteins have become a popular area of research to characterize the functional and structural properties of proteins. Protein production is traditionally done in cell-based systems such as E. coli and yeast cells; however, these systems are limited to the production of non-toxic proteins, often degrade the targeted protein through proteolysis, and consume a large amount of reagents and time. An alternative method, cell-free protein synthesis (CFPS), eliminates these issues by operating without an intact cell. Instead, protein production is done using cell lysate containing the machinery necessary for transcription and translation.
CFPS systems have been studied in several forms including the conventional batch, continuous-flow cell-free (CFCF), and continuous-exchange cell-free (CECF) formats. The CECF format, which involves the passive exchange of substrates and byproducts through a nanoporous membrane separating the transcriptional and translational machinery from the nutrients and energy necessary to support protein expression, generally has prolonged protein synthesis with the greatest protein expression yields of the three CFPS formats.
In this research proposal, we aim to optimize protein expression and increase expression yields in a CECF format device by altering the orientation of the nanoporous membrane from horizontal to vertical, in reference to the table surface. Preliminary results indicate that in this orientation, protein expression yield is increased by more than two-fold and reaction time is approximately halved due to synthesized protein settling against the bottom of the device instead of the membrane, reducing or eliminating clogging of the membrane pores.
Although this device can be applied to high-throughput protein expression, we aim to apply this device to the expression of tissue plasminogen activator (tPA), a glycoprotein involved in the breakdown of blood clots and used in stroke treatment, using wheat germ or E. coli lysate. Cell-free expression of tPA has thus far been limited to the batch format with rabbit reticulocyte lysate as a result of tPA’s complex and glycosylated structure. By synthesizing tPA using wheat germ or E. coli extract, high yields of functional tPA can be rapidly expressed within a few hours. With the validation of tPA expression in the vertical CECF device, we will then be equipped to design and fabricate an on-site CFPS device using wheat germ or E. coli lysate for the reconfigurable expression of multiple protein-based drugs. Such a device can be applied to medical facilities in military front lines and resource-limited regions of the world for portable drug expression when the need for protein-based drugs arises.