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Changing the norms: cell-free protein expression in Vibrio natriegens

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Posted April 29, 2019
This news or article is intended for readers with certain scientific or professional knowledge in the field.

Heterologous protein expression is a routine process in many labs, be it for basic research or for industrial applications. Historically, proteins are expressed in microbes optimized for this task. Different hosts are better suited for different proteins, and Escherichia coli is often the organism of choice (especially when the protein comes from a prokaryotic organism or when no post-translational modifications are required). In all cases, heterologous protein expression is a complicated process, requiring the genetic transformation of the producing cell, cellular growth, and harvesting of the expressed protein.

However, a cell is not necessary for a protein to be produced. An in vitro system containing amino acids, ribosomes, mRNA, tRNAs, and any required cofactors could do the job.The use of cell-free expression systems comes with advantages and disadvantages: usually they achieve slower production rate than cell-based systems, as the protein synthesis components are more dilute in the tube as compared to within a cell. However, cell-free systems are fantastic when it comes to the expression of toxic and aggregating proteins, while the lack of proteases and inhibitors in the mixture can improve protein production and stability. Moreover, in in particular interest for synthetic biology, cell-free systems can be adapted for multiplexing. It is possible to produce multiple protein variants to be tested without the need of laboriously generating separate bacterial strains contain each of the protein design.

 

Escherichia coli: Scanning electron micrograph of Escherichia coli, grown in culture and adhered to a cover slip. Credit: Rocky Mountain Laboratories, NIAID, Public Domain

Escherichia coli: Scanning electron micrograph of Escherichia coli, grown in culture and adhered to a cover slip. Credit: Rocky Mountain Laboratories, NIAID, Public Domain

The simplest way of performing cell-free synthesis is by using lysed cell extracts. Most methods reported use cell extracts from the model platforms E. coliSaccharomyces cerevisiae, or CHO cells. While using a familiar system is understandable, this practise may impose limitations. There are plenty of organisms and some of them may contain a more suitable transcription system for certain applications. One of these microbes is Vibrio natriegens.

V. natriegens has the fastest bacterial growth ever reported, with a doubling time of around 10 minutes. The biotechnological potential of this super-fast critter has been largely unexplored, though recently it has gained significant interest as a synthetic biology chassis. Last year’s iGEM grand prize winners worked on optimizing V. natriegens for synbio applications. George Church’s lab published a functional genomics characterisation, pinpointing its essential genes as well as the ones contributing to its fast growth. One thing is certain: since V. natriegens grows so fast, its protein synthesis machinery has to keep pace. As a result, a cell extract of V. natriegens could be an advantageous component of an improved cell-free protein expression methodology.

The use of V. natriegens extracts for cell-free synthesis has been indeed tried, where researcher have the the production of eYFP and superfolder GFP in high concentrations within a few hours. More importantly, as shown in the recent video protocol published in JoVE, the cell extracts can be prepared with low cost and used in 10 μL batch reactions in 96- or 384-well plates. Even though the productions is on the same level as E. coli extracts, the system is newer and there’s probably room for improvement.

As synthetic biology gains popularity and more researchers try to integrate it in their methodology, the “traditional” techniques start displaying their limitations. Therefore, I expect to see more out-of-the box thinking and new methodologies with specific advantages. Cell-free synthetic biology is gaining traction, while a potential replacement of E. coli as the main genetic engineering and synthetic biology workhorse will be indeed a paradigm shift. Synthetic biology embraces innovation, so experimental practices may change drastically in the years to come.

Source: PLOS EveryONE

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