Outside-the-Cell Thinking Simplifies Assembly of Pure Protein Synthesis Machinery

Never mind outside-the-box thinking. Outside-the-cell thinking promises to solve a difficult problem – how to reconstitute cellular reactions outside of biological systems, and to do so in a practical, cost-efficient way. At present, it is difficult to assemble synthetic cell-free systems that can emulate the multistep processes that occur naturally within cells.

These cell-free systems typically require multiple proteins, which may be extracted from whole cells and used directly for in vitro translation. Unfortunately, proteins extracted by this method can contain cytoplasm and other elements of the original cell – impurities that are undesirable for some applications. Another method involves purifying all the necessary proteins separately before blending them together. The separate expression and purification of the individual proteins is expensive and time consuming, however, making the production of more than several proteins at once extremely challenging.

The problems encountered with existing approaches may be overcome with an approach recently developed by scientists based at the University of California, Davis. These scientists, led by Cheemeng Tan, Ph.D., a professor of biomedical engineering, have demonstrated that bacterial consortia can be used to reconstitute cellular pathways ex vivo. The scientists applied their approach to a particularly difficult problem: the preparation of the 34 proteins involved in transcription and translation.

Pure Protein Synthesis Machinery

Details of the work accomplished in the Tan’s lab appeared in the journal Nature Chemical Biology, in an article entitled “Synthetic Microbial Consortia Enable Rapid Assembly of Pure Translation Machinery.” The article presents an approach that could reduce the time and cost associated with preparing multiprotein systems that emulate cellular processes.

“Here, we engineer synthetic microbial consortia consisting of between 15 and 34 Escherichia coli strains to assemble the 34 proteins in a single culturing, lysis, and purification procedure,” wrote the article’s authors. “The expression of these proteins is controlled by synthetic genetic modules to produce the proteins at the correct ratios.”

Essentially, the Tan lab circumvented the limitations of existing techniques by synthetically engineering strains of E. coli bacteria to produce the required proteins of correct quantity within a single mixed culture. By manipulating transcription rates, translation rates, and relative strain densities, the lab found that it could induce the bacterial consortia to produce correct quantities of the translation machinery.

“I believe the work will open doors to fundamental improvement in the protein yield of pure cell-free transcription–translation systems and throughput of studying disease-relevant pathways outside of living cells,” asserted Dr. Tan.

The Tan team calls its method TraMOS, for Translation Machinery One Shot. In the current study, TraMOS-produced proteins were used in a test that screens for the presence of peptides that inhibit a protease. Because proteases are commonly involved in the life cycle of parasites and cancer development, a test that could locate and identify many of the protease inhibitors all at once will be useful for drug development.

The new approach to reconstitute cellular machinery could have other applications. Rapid yet high-purity reconstitution of the cellular reactions is critical for the high-throughput study of cellular pathways and cell-free diagnostic tests for various diseases.

By reducing the time and cost associated with preparing multiprotein systems, the Tan lab’s approach enables high-throughput applications of TraMOS without having to invest in additional purification equipment. Unlike existing approaches, scientists can customize the expression and control of proteins using the TraMOS approach. Most labs that routinely perform protein purification already have the equipment to use the TraMOS approach, making it easy to implement and democratizing access to the system. The microbial consortia-based approach may be generalized for the synthesis of other multiprotein systems, making it a potential game changer for high-throughput cell-free applications.