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Expanding the scope of cell-free protein expression

Alternative robust cell-free expression system based on a marine bacterium could facilitate advances in synthetic biology and basic research

By Benjamin Boettner

The use of the microbial host E. coli has been crucial for the development of many important biotechnologies. For example, E. coli, has been used as a living factory in the large-scale production of valuable therapeutic proteins and other important biomolecules. Because in vivo bacterial culture is often a lengthy and expensive procedure, especially at large scales or in the parallel synthesis of many different proteins at the same time, researchers have developed in vitro cell-free expressions systems to do the same work in a fraction of the time and at much lower costs.

In this time-lapse video, the Vibrio natriegens cell-free extract is used to express a luciferase enzyme, whose luminescence increases following addition of a substrate and then drops again within 5 minutes of the reaction. Credit: Wyss Institute at Harvard University

Microbial in vitro cell-free expression systems harness the molecular machinery of bacteria such as E. coli as crude or purified extracts to perform the complex task of translating a user-specified DNA template into its protein product. In addition, because cell-free expression is not limited by the strict conditions needed for optimal bacterial growth in vivo, researchers are able to produce and characterize proteins or other biomolecules that they may not have access to otherwise.

Yet, despite the overall potential of E. coli as a source for cell-free expression systems at different scales, researchers have recognized that other bacterial species could provide unique advantages such as specialized gene expression programs that evolved to adjust their metabolism to specific environments, or simply faster growth rates. Being able to harness their molecular machineries as well could open up new basic and biotechnological research applications.

A research team led by founding and Core Faculty member George Church at the Wyss Institute has developed a low-cost and highly efficient alternative to E. coli as a bacterial cell-free expression system by leveraging Vibrio natriegens, a salt-loving (halophile) marine bacterium that in less than 10 minutes divides faster than any other known organism in media containing inexpensive nutrients. The team’s new system can be generated more rapidly and cost-effectively on-site and could be used for a wide range of applications, including the parallel expression of multiple proteins, engineering of metabolic pathways that produce valuable chemicals, and the design of artificial genetic circuitry for synthetic biology applications. The advance is reported in ACS Synthetic Biology.

Microbial cell-free expression systems are generated by growing bacterial cultures to a certain density, rupturing of the bacteria, and removing unwanted cellular debris. Left in the remaining crude extract are all necessary elements for reading DNA into messenger RNA (mRNA) intermediates (transcription) and translating those into functional proteins (translation), as well as for protein folding and energy metabolism. After adding in a DNA template encoding a specific protein, the basic building blocks of mRNA intermediates and proteins, an energy source, as well as additional cofactors and salts, a succession of reactions is started that leads to the exclusive synthesis of the protein product according to the template’s instructions.

The team started engineering their V. natriegens cell-free expression system applying conditions commonly used for E. coli. “Using a fluorescent model protein as a read-out, we tweaked the parameters important for growth of the bacteria and the cell-free expression reaction itself. This approach enabled us to prepare reaction-ready extracts that can express the model protein at a sustained high level over long periods of time,” said the study’s first author Daniel Wiegand, a Research Scientist at the Wyss Institute. “Our head-to-head comparisons show that our optimized system from wild-type V. natriegens performs as good as a highly engineered strain of E. coli.”

This new cell-free expression system with its unique features and high cost-effectiveness could be used as a test bed in multiple basic research and synthetic biology efforts, including the identification of designer enzymes with altered or boosted activities from thousands of candidates.

George Church

However, equivalent approaches with E. coli require significantly longer times to prepare reaction-ready extracts, and commercially available E. coli extracts for cell-free protein expression are significantly more expensive than the team’s V. natriegens-based extracts, which can be made for merely $1 per reaction.

“This new cell-free expression system with its unique features and high cost-effectiveness could be used as a test bed in multiple basic research and synthetic biology efforts, including the identification of designer enzymes with altered or boosted activities from thousands of candidates, for example DNA polymerase molecule variants that can more efficiently read or write DNA,” said Church, Ph.D., who also is Professor of Genetics at Harvard Medical School (HMS) and of Health Sciences and Technology at Harvard University and the Massachusetts Institute of Technology (MIT). “It could also become a new discovery tool in metabolic engineering approaches to produce coveted chemicals or be used in strategies aiming to synthesize therapeutic proteins.”

Additional authors on the study are postdoctoral fellow Henry Lee, Ph.D., and postdoctoral fellow and co-corresponding author Nili Ostrov, Ph.D., two members of Church’s team. The work was funded by grants from the National Institute of General Medical Sciences at the National Institutes of Health and the Department Energy.

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