Ribosome Pegging: An Antibiotic-free System to Produce Recombinant Proteins

Application

Antibiotic-free method to prevent the growth of cells that fail to express high levels of a non-essential protein (target protein).

Key Benefits

Prevents the use of antibiotics and markers that promote resistance.
Facilitates the deployment of recombinant bacteria in uncontrolled environments.
Stabilizes heterologous expression of a non-essential protein at high levels.
Can be applied to any transformable organism.

Market Summary

Recombinant protein expression systems allow you to obtain large amounts of commercially and/or pharmaceutically relevant protein such as Human Insulin, used for the treatment of diabetes. Recombinant proteins are generally expressed using bacterial systems, yeasts, transgenic plants, mammalian cells and insect cells. However, the burden of overexpressing a non-essential protein for the cells makes it very difficult to maintain constitutive and reliable high levels of expression. To maintain the existence of foreign DNA in host cells, antibiotic or other selective agents are used, which increase the overall bioprocess cost and generate environmental concerns. Therefore, there is a need to develop methods to maximize expression of foreign genes within industrial fermenters free of antibiotics or other selective agents.

Technical Summary

Emory researchers developed a system using cells defective in essential ribosomal protein L23, where they co-express this ribosomal gene with the foreign gene. The concept behind the system is that if the mutant cells fail to express the foreign gene, then they don’t grow well. Basically, the expression of a foreign gene of interest is "pegged" to one that is essential in any environment and highly expressed and, since all organisms express dozens of essential ribosomal proteins at high levels it should be possible to "peg" dozens of foreign genes within a single genetically modified cell. This system was successfully used to express the commercially relevant enzymes Organophosphate insecticide malathion, Agrobacterium radiobacter phosphotriesterase (arPTE), and Pseudomonas oleovorans organophosphate hydrolase (PoOph) in Escherichia coli. Additionally, in vitro experiments showed that using ribosome pegging is possible to obtain significantly more recombinant Red Fluorescent Protein (RFP) than using the pET system, the most commonly used bacterial platform for the overexpression of genes.