What Is Cell Free Protein Synthesis

Template + Cell Extract = Protein Synthesis

An introduction to Cell-free protein synthesis

Two basic components are needed to accomplish in vitro protein expression: the genetic template encoding the target protein and a reaction solution containing the necessary transcriptional and translational molecular machinery. Cell extracts supply all or most of the molecules of the reaction solution, including:

  • RNA polymerases for mRNA transcription
  • ribosomes for polypeptide translation
  • enzymatic cofactors and an energy source
  • cellular components essential for proper protein folding

Cell lysates provide the correct composition and proportion of enzymes and building blocks required for translation. Cell membranes are removed to leave only the cytosolic and organelle components of the cell . The first types of lysates developed for cell-free protein expression were derived from prokaryotic organisms. More recently, systems based on extracts from insect cells, mammalian cells and human cells have been developed and made commercially available.

Study Of Protein Expression Translation And Folding

The PURE system contains the minimal set of factors necessary for in vitro protein translation. It is largely free of chaperones and other cellular factors for post-translational modifications, thus providing a starting point to study the involvement of these factors in transcription/translation regulation and nascent chain folding .

It can also be used to produce clean proteins which, if purified from traditional cellular hosts, may come with undesired modifications or bound co-factors. A number of research labs studying translation routinely use home-made reconstituted systems to study different aspects of translation.

Applications For Which Cell

  • Experiments to characterize proteinprotein interactions and proteinnucleic acid interactions
  • Rapid and high-throughput expression of mutant or truncated proteins for functional analysis
  • Expression of mammalian proteins with proper glycosylation and native post-translational modifications
  • Labeling of proteins with stable isotopes for structural analysis
  • Production of functional virons or toxic polypeptides
  • Analysis of components required for protein folding, protein stability or protein degradation

The diagram provides an example of application of a cell-free protein expression method combined with protein mass spectrometry .

Workflow for heavy recombinant protein expression, purification and mass spectrometry analysis. Cell lysates are combined with the reaction mixture, vector DNA and either heavy or light stable isotopelabeled amino acids to express recombinant proteins. Expressed proteins are then purified and digested into peptides for LC-MS analysis. This method enables the quantitation of stable isotopelabeled proteins.

This 118-page handbook provides comprehensive information about protein expression and will help you choose the right expression system and purification technologies for your specific application and needs. Get tips and tricks when starting an experiment, and find answers to everyday problems related to protein expression.

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The Classic Batch Reaction Method

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Protein production has been done in vivo since its induction in 1976. This requires the insertion of genetic material called a plasmid into a cells DNA. Plasmids are circular snippets of DNA that cells naturally take up and institute into their genetic code for more diversity. Scientists have hijacked this phenomenon by creating synthetic plasmids called vectors which are inserted into cells via heat shock or electroporation. Cells that take up the vector are separated and grown into large colonies. When the colonies reach a sufficient size, they are induced to produce the target protein in abundance by activating the inserted gene. These colonies are lysed and the cellular extract is gathered. The extract contains a variety of contaminants including many proteins aside from the target, so it must be purified a process so involved, we wrote an entire blog about it: Protein Purification. The Classic Method requires a large initial time commitment to work out a viable protocol for each step. Even after the protocol is in place, the operation may be hampered by difficult uptake, slow cell growth, toxic proteins, poor yields, etc.

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Incorporation Of Unnatural Amino Acids Into Proteins

The translational machinery, including aminoacyl-tRNA synthetases, can tolerate a wide range of unnatural amino acids, allowing the genetic code to be expanded or even reassigned . One of the earliest applications of cell-free protein synthesis was the incorporation of unnatural amino acids into a specific position of an in vitro synthesized protein. Schultz and colleagues first demonstrated the incorporation of unnatural amino acids into an amber stop codon that substituted a sense codon in a protein in an E. coli extract . It took more than a decade for the Schultz laboratory to engineer living cells to allow such expansion of the genetic code in vivo . And still, compared to cell-based approaches , cell-free systems are easier to manipulate for incorporation of unnatural amino acids. The reconstituted cell-free systems are especially suited for these applications . Using the reconstituted cell-free system, researchers can simply remove the competing factors, such as release factors, tRNAs or aminoacyl-tRNA synthetases, or replace them with an tRNA-synthetase pair from another organism, or an engineered aminoacyl-tRNA synthetase . Moreover, tRNAs can be pre-acylated with unnatural amino acids using natural enzymes or engineered ribozymes . The combined use of pre-charged tRNAs and the reconstituted cell-free system has allowed unnatural peptides or peptidomimetics containing mostly unnatural amino acids to be synthesized for potential applications in therapeutics .

So What Are The Advantages Of Cell Free Protein Synthesis As Opposed To Synthesising In Vivo

Here are a few, taken from reference 1:1. Improved protein yields: Since there are no cellular processes going on so virtually all of the resources available in the extract go towards the production of your protein of interest.

2. An optimal environment for the production of your protein. An open system, with no cell walls . You can add components to create the most optimal environment for your protein production.

3. No cell viability concerns if the protein expressed is toxic. Since no cell growth is needed.

You can synthesis your protein in vitro either from a suitably constructed DNA template using a coupled system that will perform in vitro trancription and translation in tandem , or you can do the two steps separately. Either way you can make lots of protein made in just a few hours. There are number of companies that offer invitro coupled systems and invitro transcription or in vitro translations systems.

Transcription is done using the phage RNA polymerases T3, T7 or SP6 so the coding sequence you want to express must be flanked by the promoters for one of these polymerases. In vitro transcription kits contain the polymerase ribonucleotides, buffers and magnesium required for transcription.

Typically, E.Coli S30 fraction is used for prokaryotic expression 554-561).

Rabbit Reticulocyte lysate and wheat germ extract are the most popular lysates for eukaryotic expression.

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Inhibitors Of Amino Acid Metabolism

In the cell-free protein synthesis method, most of the stable isotope scrambling is negligible. However, non-labile 2H dilution with 1H still occurs in the E. coli cell-free system. Furthermore, 15N scrambling and dilution also remain among aspartate, asparagine, glutamate, and glutamine in the E. coli cell-free system. For such problems, Yokoyama, Kigawa, and their colleagues reported the successful use of chemical inhibitors of the metabolic enzymes, which prevented the scrambling and dilution of the stable isotope . The non-labile 2H dilutions at the α- and β-positions in certain amino acids are catalyzed by pyridoxal 5â²-phosphate -requiring enzymes, in the ordinary cell-free system based on 1H2O. Therefore, the cell-free synthesis of perdeuterated proteins from perdeuterated amino acids was achieved by the cell-free protein synthesis reaction in which all of the reagents, including the E. coli cell extract, were prepared with 2H2O . However, for all such amino acids, except methionine and cysteine, the non-labile 2H dilution can be suppressed by using two PLP-requiring enzyme inhibitors , aminooxyacetate and d-malate , in the cell-free reaction. Therefore, this technique enables the cell-free synthesis of perdeuterated proteins from perdeuterated amino acids with the 1H2O-based reaction .

Tatsuya Sawasaki, … Yaeta Endo, in, 2001

Development And Application Of Cfps Models

Introduction to the NEBExpress® Cell-free Protein Synthesis System

Continuous models are typically used to simulate CFPS systems. They make use of ordinary differential equations and algebraic equations to dynamically describe model states. In a structured model, equations incorporate affinity constants and other parameters. To reduce complexity, models can be formulated following an unstructured black-box approach, considering only apparent kinetics . The differences between the model types are dynamic. Often, models are partly structured to focus on selected segments of the reaction network with particular interest. We call these approaches hybrid models. The quality of CFPS mechanistic models relies heavily on the proper model structure and the correct identification of model parameters. Given the complex nature of CFPS, the precondition of independent datasets for parameter identification is challenging and may require repeated careful consideration for each regression analysis . Stochastic effects play only a minor role in most of the classical CFPS modeling approaches. In liposome or droplet-based CFPS, due to small reaction volumes, low numbers of molecules may cause rendering reactions between different molecules in stochastic events. Under such conditions, a description with a discrete and stochastic model is preferable .

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Ribosome And Polysome Profile Analysis

Cell-free extract samples were placed in 520% sucrose gradients with 10mM Tris-HCL, pH 8.0, 12mM magnesium acetate, 100mM NH4CL, and 1mM DTT. Cell-free reaction samples were quenched in liquid nitrogen and placed in 1040% sucrose gradients. All samples were centrifuged in an SW41 Ti rotor at 30,000rpm for 3h at 4°C. The gradients were analyzed by pumping them through a UV cuvette and measuring their absorbance at 260nm.

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Esterase Activity Staining On Polyacrylamide Gel

The protein esterase 2 in the CFPS solution was separated by SDS-polyacrylamide gel in a 100 mM Tris-HCl . After electrophoresis and EST2 renaturation by removing SDS, the gel was mixed with the substrate and the product was linked with Fast Blue BB for the formation of an insoluble color complex, indicating the presence and amount of EST2 . Subsequently, the other proteins in the gels were strained by the Coomassie blue dye .

Batch Continuous Flow And Continuous Exchange Formats

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CFPS reactions can be performed in batch format for simplified setup, or in continuous formats for improved protein yields. Reactions are most easily, quickly, and cheaply set up in batch format because all necessary reactants are added to a single tube and incubated for protein synthesis to occur . However, the duration of a batch reaction is dependent on the amount of substrate available and the amount of inhibitory byproduct produced, which can result in low yields for some platforms . On the other hand, continuous flow and continuous exchange CFPS reactions utilize a two-chamber system to supply reactants and remove products, for increased reaction duration and higher protein yields . In continuous exchange cell-free , the CFPS reaction is separated from a reactant-rich feed solution via a semi-permeable membrane, such that new reactants move into the reaction and byproducts move out, while the protein product remains in the reaction compartment . For continuous flow cell-free , the feed solution is continuously pumped into the reaction chamber, while the protein of interest and other byproducts are pushed out through an ultrafiltration membrane .

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The Dependence Of Cell

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      A stable cell-free system has been obtained from E. coli which incorporates C14-valine into protein at a rapid rate. It was shown that this apparent protein synthesis was energy-dependent, was stimulated by a mixture of L-amino acids, and was markedly inhibited by RNAase, puromycin, and chloramphenicol. The present communication describes a novel characteristic of the system, that is, a requirement for template RNA, needed for amino acid incorporation even in the presence of soluble RNA and ribosomes. It will also be shown that the amino acid incorporation stimulated by the addition of template RNA has many properties expected of de novo protein synthesis. Naturally occurring RNA as well as a synthetic polynucleotide were active in this system. The synthetic polynucleotide appears to contain the code for the synthesis of a protein containing only one amino acid. Part of these data have been presented in preliminary reports.,

      Filaggrin Expression In Vivo

      After achieving a high-yield FLG synthesis via optimizing the CFPS system in this study, we next explore the FLG synthesis in vivo. The CFPS system has been compared with in vivo recombinant protein synthesis platform in terms of usability, flexibility, scalability, and, more importantly, productivity. One of the features is that the CFPS is accomplished in a single reaction, whereas the in vivo system requires multiple procedures for protein expression . This allowed the CFPS to become an essential tool to rapidly screen and eliminate factors limiting the production of target proteins. For example, we identify that the codon bias was the main limiting factor in FLG expression without conducting time-consuming steps of cloning, transformation, and cell cultivation. This highlights the versatility of cell-free platform as a protein production optimization toolkit. Additionally, the CFPS system does not require host strain modification to accommodate the exogenous DNA to express target protein, which causes the poor growth of the host, protein inactivity, and low protein yields . Not only the exogenous protein expression but also the presence of the exogenous gene in bacteria can be a burden to the host cell .

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      Coupled And Uncoupled Formats

      CFPS reactions can be performed in coupled or uncoupled formats, and the choice is dependent on the platform being used and the users needs. Coupled reactions allow transcription and translation to take place within a single tube, such that the supplied DNA template can be transcribed into mRNA, which is then translated into protein within a one-pot reaction. The advantage of coupled CFPS is the ease of reaction setup, but it may result in suboptimal yields for eukaryotic platforms. Uncoupled reactions typically consist of an in vitro transcription reaction followed by mRNA purification the purified transcripts are then supplied to the cell-free translation reaction containing the cell extract for production of the protein of interest. Uncoupled reactions are more often utilized in eukaryotic CFPS platforms due to mRNA processing for more efficient translation of certain transcripts. As an example, pseudouridine modification for mRNA in the rabbit reticulocyte platform has been demonstrated to enhance translation . Uncoupled reactions also allow for different conditions between transcription and translation reactions, which can improve yields . Uncoupled reactions can be achieved in any platform by supplying the reaction with mRNA instead of DNA, but mRNA can be more difficult to handle and does degrade more quickly in the CPFS reaction .

      Introduction To In Vitro Protein Expression

      Synthesis of membrane proteins using cell-free system (Extract version)

      In vitro protein expression is a technique that enables researchers to rapidly express and manufacture small amounts of functional proteins. Compared to in vivo techniques based on bacterial or tissue culture cells, in vitro protein expression is considerably faster because it does not require gene transfection, cell culture or extensive protein purification.

      Although in vitro expression is not practical for commercial large-scale recombinant protein production, it has a variety of features that make it considerably more useful and flexible for many research applications.

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      Host Selection And Codon Optimization

      Improving Protein Synthesis Using Rare tRNA Expressed Cell Extract

      Since the transcription and translation apparatus is well conserved in the cell extract, host strain selection can revamp the overall protein synthesis rate in the CFPS system and likewise in in vivo recombinant protein synthesis platforms. Although the genetic code is universally shared across species, different codon usage biases strongly correlate with protein synthesis between the host strain and the origin of a gene of interest . This codon usage impacts protein expression levels and folding efficiency during the translation . As the abundance of tRNA at the cellular level speeds up ribosomes to decode a codon , codon optimization and rare tRNA supplementation can improve transcription and translation efficiency without a shortage of tRNA availability. Therefore, choosing an appropriate Escherichia coli host that supports sufficient rare tRNA expression provides a decisive improvement in protein production yields, particularly in a prokaryotic heterologous gene expression system for a codon biased exotic gene such as FLG.

      Introduction To Platform Categorization

      In the 60 years since cell-free protein synthesis emerged, a multitude of platforms have been developed based on cell extracts from a variety of organisms. These include extracts from bacterial, archaeal, plant, mammalian, and human cell lines. Each resulting platform varies in ease of preparation, protein yields, and in possible applications resulting from the unique biochemistry of the given organism. In this review, we have divided these various platforms into two categories: high adoption and low adoption . The platforms have been categorized based on our understanding of their development and the degree to which they have been adopted by the field, as quantified by the number of peer-reviewed publications that utilize each platform . This categorization allows new users to identify platforms that have been best established and to explore the applications that they enable. We believe that the depth of literature available for these platforms makes them optimally suited for newer users. Low adoption platforms may be particularly useful for niche applications, but have not been optimized thoroughly, or are currently emerging in the field. Therefore, these platforms may be more difficult to implement due to minimal development. Platforms with fewer than 25 peer-reviewed publications to date have been categorized as low adoption.

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