Improving the genetic circuits in eukaryotic protein production systems—like human cell lines or Chinese hamster ovaries—has the potential to enhance their value so they may offer not only their own inherent benefits, but also the higher protein production and lower production costs associated with such prokaryotic systems as Escherichia coli.
The protein-responsive upregulating riboswitches (ON-riboswitches) developed for a wheat-germ extract production platform by scientists at the Proteo-Science Center (PROS) at Japan’s Ehime University are a step towards doing that. These ON-riboswitches can create the complex genetic circuits needed for multistep gene regulatory cascades.
“Our goal is to create programmed cell-free systems or artificial cells with functions that not only are similar to those of natural cells, but that surpass them,” Atsushi Ogawa, PhD, associate professor at PROS, and first author of a recent paper, tells GEN. “Our riboswitches can be engineered to respond to user-defined ligands,” so they may meet the needs of some biopharmaceutical manufacturers.
Eukaryotic production systems have some distinct advantages that prokaryotic systems lack. Notably, they include “higher compatibility with eukaryotic proteins and higher functionality around ambient temperatures,” Ogawa points out. They are limited, though, by low productivity and higher production costs when compared to E. coli systems.
The widely used E. coli protein production systems, for their part, offer high yield, fast and easy cultivation, and are cost-efficient and easy to engineer. However, proteins produced this way may not fold correctly, and post-translational modifications are limited. E. coli systems also lack the endogenous membrane structures needed to synthesize certain membrane proteins.
There is a solid case, therefore, for improving eukaryotic protein production systems.
Four switches created
Hybridization switches are a key feature of the ON-riboswitches the team designed. After they were added, the designs “upregulated expression to 20-fold through self-cleavage by a hammerhead ribozyme in response to the corresponding protein ligands expressed in situ,” the scientists report.
“Hybridization switches aren’t always necessary for hammerhead hybridization-based ON-riboswitches,” Ogawa stresses. “They are required when small, protein-biding aptamers, such as CS1 and CS2, are used.”
The next iteration combined those ON-riboswitches with similar hammerhead ribozyme ON-riboswitches that responded to small molecules to regulate protein-ligand expression. Because the four types of ON-riboswitches they designed, although similar, could not interact with one another, they were able to regulate two-step cascades simultaneously. Three-step cascades also were created.
“Interestingly,” the scientists note, “the switching efficiency of each multistep cascade constructed was equivalent to that of the worst step within it.” This suggests more complex cascades with additional switches may be constructed from other orthogonal protein-responsive ON-riboswitches without sacrificing production efficiency.
Next, the “multistep, but simple, gene regulatory cascades” the researchers created will be combined with “OFF-riboswitches and other regulators to create more intricate circuits, including various types of logic gates,” Ogawa says. Ultimately, these genetic switches could play an important role in creating complex eukaryotic genetic circuits for programmed cell-free systems or artificial cells.
