A User's Guide to Cell-Free Protein Synthesis

Abstract

Cell-free protein synthesis (CFPS) is a platform technology that provides new opportunities for protein expression, metabolic engineering, therapeutic development, education, and more. The advantages of CFPS over in vivo protein expression include its open system, the elimination of reliance on living cells, and the ability to focus all system energy on production of the protein of interest. Over the last 60 years, the CFPS platform has grown and diversified greatly, and it continues to evolve today. Both new applications and new types of extracts based on a variety of organisms are current areas of development. However, new users interested in CFPS may find it challenging to implement a cell-free platform in their laboratory due to the technical and functional considerations involved in choosing and executing a platform that best suits their needs. Here we hope to reduce this barrier to implementing CFPS by clarifying the similarities and differences amongst cell-free platforms, highlighting the various applications that have been accomplished in each of them, and detailing the main methodological and instrumental requirement for their preparation. Additionally, this review will help to contextualize the landscape of work that has been done using CFPS and showcase the diversity of applications that it enables.

Keywords: cell-free metabolic engineering (CFME); cell-free protein expression (CFPE); cell-free protein synthesis (CFPS); cell-free synthetic biology; in vitro protein synthesis; in vitro transcription-translation (TX-TL).

Figure 1

A comparison of cell-free and in vivo protein synthesis methods. Through visualization of the main steps of in vitro and in vivo protein expression, the advantages of cell-free protein synthesis emerge. These include the elimination of the transformation step, an open reaction for direct manipulation of the environment of protein production, the lack of constraints based on the cell’s life objectives, the channeling of all energy toward production of the protein of interest, and the ability to store extracts for on-demand protein expression. Green cylinders represent synthesized green fluorescent protein (GFP).

Figure 2

Comparison of protein yields across cell-free platforms. The volumetric yield of each platform is reported for batch reactions producing GFP. Platforms that report volumetric yield for reporter proteins luciferase (*) or ChiAΔ4 (**) are indicated. Information for batch mode protein yields of the Arabidopsis and Neurospora crassa platforms was not found. Yields were obtained from the following sources: E. coli [8], wheat germ [9], Vibrio natriegens [10], Leishmania tarentolae [11], tobacco [12], HeLa [13], Pseudomonas putida [14], Streptomyces [15], Bacillus megaterium [16], Chinese hamster ovary [17], insect [18], Bacillus subtilis [16], yeast [19], archaeal [20], and rabbit reticulocyte [21].

Figure 3

Comparison of batch, continuous flow, and continuous exchange reaction formats. Batch reactions contain all the necessary reactants within a single reaction vessel. Continuous exchange formats utilize a dialysis membrane that allows reactants to move into the reaction and byproducts to move out, while the protein of interest remains in the reaction compartment. Continuous flow formats allow a feed solution to be continuously pumped into the reaction chamber while the protein of interest and other reaction byproducts are filtered out of the reaction.

Figure 4

High adoption cell-free platforms and their applications. (A) Web of the applications enabled by high adoption cell-free platforms. The connections shown are based on applications that have been published for each respective platform. Applications under “difficult to synthesize proteins” include the production of antibodies, large proteins, ice structuring proteins, and metalloproteins. Miscellaneous applications include studies of translational machinery, genetic circuits, metabolic engineering, and genetic code expansion. (B) Cumulative number of peer-reviewed publications over the last 60 years for high adoption platforms. The metric of cumulative publications by platform is used to indicate which platforms are most utilized, with platforms having over 25 papers categorized as high adoption. These data were generated by totaling papers from a PubMed Boolean search of the following: (“cell free protein synthesis” OR “in vitro transcription translation” OR “in vitro protein synthesis” OR “cell free protein expression” OR “tx tl” OR “cell-free translation”) AND “platform name.” The platform name used for each search corresponds to the name listed in the graph’s key. This information was collected on 23 December 2018, and the search results for each platform can be found in Supplemental Information. While this metric is an indicator of the level of adoption for each platform, it does suffer from false positive search results, such as papers reporting studies in which the researchers produce recombinant proteins from the organism of interest rather than from cell extract derived from that organism.

Figure 5

Low adoption cell-free platforms and their applications. (A) Web of the applications enabled by low adoption cell-free platforms. Connections shown are based on applications that have been published or that have been proposed in publications. Applications under “difficult to synthesize proteins” include high GC content proteins, antimicrobial peptides, pharmaceutical proteins, and thermophilic proteins. Miscellaneous applications include studies of translational machinery, investigation of antibiotic resistance, genetic circuits, metabolic engineering, and genetic code expansion. (B) Cumulative number of peer-reviewed publications over the last 60 years for low adoption platforms. We have used the metric of cumulative publications to indicate which platforms are less utilized and have categorized platforms with under 25 papers as low adoption platforms. These data were generated by totaling papers from a PubMed Boolean search of the following: (“cell free protein synthesis” OR “in vitro transcription translation” OR “in vitro protein synthesis” OR “cell free protein expression” OR “tx tl” OR “cell-free translation”) AND “platform name.” The platform name used for each search corresponds to the name listed in the graph’s key. While this metric is an indicator of the level of adoption for each platform, it does suffer from inconsistencies due to irrelevant search results, such as papers reporting studies in which the researchers produce proteins from the organism of interest rather than from cell extract derived from the organism. This inconsistency was significant for platforms with fewer papers, so we pursued data curation to remove irrelevant papers and add in missing papers. This information was collected on 23 December 2018, and curated search results for each platform can be found in Supplemental Information, where red indicates that the paper was removed from the search results and green indicates that the paper was added to the search results.

Figure 6

General workflow for preparation of cell-free extract and set up of CFPS reactions. A visualization from cell growth to the CFPS reaction is depicted above for a new user, highlighting the main steps involved.