Advancing Cell-Free Technologies

Improving productivity and product diversity for CFPS

Increasing productivity. Cell-free protein synthesis is now being used for producing a wide variety of proteins. Platforms are now available based on human cell extracts and plant cell extracts as well as bacterial. Further, significant progress has been reported by the DeLisa and Jewett labs toward producing glycosylated proteins. While some increases in system volumetric productivity have also been reported, we believe that a significant opportunity for major improvements still exists. This belief is supported by several observations from our lab. The most significant resulted from using data obtained by examining the impact of individually overexpressing nearly 4000 E.coli genes on the expression and activation of four different proteins. A few of the most beneficial proteins were added at higher concentrations and the genes encoding four of the most detrimental proteins were deleted. When the most influential of these changes were combined with increased amino acid concentrations and pH stabilization, approximately 4 fold increases in product accumulation were achieved. Total protein accumulation as high as 5 mg/ml was reached in a 6 hr reaction period. Unfortunately, due to the complexity of this very large data set, these results were not published.

In future work, we plan to confirm and improve upon these observations. Opportunities exist in improving: energy supply, nucleotide triphosphate supply, mRNA stability, translation initiation rate, translation elongation rate, improved protein folding (discussed in more detail below), and in substrate concentration and biochemical environment stabilization.

Improving protein folding and solubility. While CFPS systems are sometimes called transcription/translation systems, the most demanding challenge is in folding the protein properly. This is particularly true for more complicated proteins of value as pharmaceuticals and also for complex enzymes, especially when cofactors must be assembled and installed and when disulfide bonds must be correctly formed. For these targets, a variety of chaperones and other helper proteins may be advantageous. To address these needs, we are developing a Design of Experiment approach to rapidly discover the individual and synergistic contributions of a variety of folding aids. In addition, we have observed significant advantages from adjusting the biophysical environment relative to, for example, the –SH/S-S redox potential and the ionic strength of the CFPS solution. Specific benefits can also be provided by adjusting concentrations of required cofactors that must be installed to activate enzymes. The access provided by the cell-free approach opens many opportunities to efficiently produce proteins that are difficult to activate during in vivo expression.

Increasing production duration. While improvements in volumetric productivity can be advantageous, additional productivity gains and cost reductions can be gained by extending the productive duration of the CFPS reactions. Past experiments have indicated significant gains from the periodic addition of additional amino acids, nucleotides, energy source molecules, and magnesium. More recent experiments have focused on maintaining the activities of metabolic components required for sustained energy supply.