This work was funded by the National Institute of General Medical Sciences 1U01GM110714-01 and Department of Energy DE-FG02-02ER63445. The authors would like to thank Dr. Richard Kohman, Dr. Jenny Tam, and Dr. Edgar Goluch for helpful advice on constructing the protocol section of this manuscript.

<ol>
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DJW, NO, and GMC have filed a patent related to this work.

This protocol has been optimized for wild-type V. natriegens and bacterial growth media comprised of LB supplemented with V2 salts (Table of Materials). Other strains of V. natriegens can be similarly cultured to generate crude cell extracts for cell-free reactions; however, their use requires additional optimization of this protocol. Additionally, this cell-free protein expression system has been optimized using 3-phosphoglyceric acid (3-PGA) as the primary energy regeneration source. Other energy regeneration sources may be used; however, optimization of reagents and calibration will likely be required to obtain high yielding protein expression10,11.
Specific attention to several critical steps in this protocol will ensure maximal extract productivity for high-yielding cell-free protein production. First, crude cell extract must be prepared from V. natriegens cell cultures harvested in a mid-exponential phase of growth; protein yield is maximal when cultures reach an OD600 = 1.0 &#177; 0.2. While cell-free protein production is possible from cells harvested at a range of optical densities, we have previously found that cells harvested in an exponential state of growth yield significantly more protein9. The observed effects of optical density on crude cell extract performance are consistent with those reported for other cell-free expression systems derived from cells grown in batch culture conditions1. Because V. natriegens grows at a rapid rate, it is critical to closely monitor cultures&#8217; optical densities. In general, it is expected that V. natriegens cultures should reproducibly reach an OD600 of 1.0 within 1&#8722;1.5 h using this protocol; however, individual growth conditions such as the use of baffled versus non-baffled flasks, which affects aeration, or air versus water incubation, which affects the rate and stability of incubation temperature, may alter growth time. Furthermore, it is generally recommended to culture at least 250 mL of V. natriegens in a 1 L baffled flask to ensure a large cell pellet at harvest for easy manipulation and transfer. This greatly improves the success of crude cell extract preparation as well as increases the total volume of extract produced from a pellet. When using smaller scale preparation, culture conditions and reagents can be adjusted appropriately. For large-scale fermentation, further optimization of culture conditions may be required. Finally, to ensure high protein yield, it is critical that cell pellets are processed immediately after harvesting, or within 1&#8722;2 days of storing at -80 &#176;C.
The proper lysis of the cell pellet by pulse sonication is critical to the success of cell-free protein expression and is often the most difficult aspect of this protocol for new users. Typically, a well lysed pellet will yield a significant volume of liquid extract that is free of debris. The extract should be slightly viscous but can be easily pipetted when aliquoting into storage tubes before flash freezing in liquid nitrogen. Figure 3 depicts a representation of a well lysed pellet (Figure 3A) in comparison to a poorly lysed pellet (Figure 3B) after the post lysis centrifugation step. A major indication of complete cell lysis is a crude cell extract total protein concentration &#62; 20 mg/mL as determined by a total protein assay (step 2.13). Over-sonication or excessive heating of the crude cell extract will damage the cellular machinery, which cannot be determined without performing a cell-free reaction. Thus, it is highly beneficial to test extract efficiency with a control reaction before dedicating significant time and effort to downstream protein expression applications. While additional optimization may be necessary for different sonication equipment, the pulse sonication steps described have been highly reproducible in our hands.
The use of linear DNA template derived from PCR amplification, restriction enzyme digest, or commercial gene synthesis can significantly increase the capacity for high-throughput and rapid protein production in cell-free expression systems24. While protein production from PCR amplified linear template has been demonstrated, the yield of these reactions are approximately 13.5-fold lower compared to reactions using plasmid DNA template at equimolar ratios9. This is primarily due to the instability of the linear DNA template which is likely degraded by endogenous nucleases present in V. natriegens crude cell extract. While lambda phage protein GamS has been previously used to protect Linear DNA template24,25, it was found to be incompatible with V. natriegens extracts9. Additionally, while increasing the concentration of linear DNA template may allow for a higher protein yield, its fast degradation in crude cell extract will still be a major problem.
A solution to overcoming linear DNA template degradation may be to supplement cell-free reactions with mRNA template generated from in vitro transcription of linear DNA. On the other hand, the use of an RNase inhibitor offers significant protection against mRNA transcript degradation and appreciable protein yields can be obtained in the 10 &#956;L cell-free reaction format (Figure 5A,B). Cloning of linear DNA into a circular template through TA ligation, TOPO cloning, Golden Gate assembly, or other recombination methods may be used to circumvent template degradation. Nevertheless, further approaches for inhibition of nuclease activity will be necessary for efficient protein expression using linear DNA template.
To date, several different approaches have been proposed for preparation of crude extract for cell-free protein expression9,10,11,26. In developing this protocol, we sought to maximize user accessibility, reduce overall cost, and minimize time-consuming steps. For example, a high protein yield is achieved using a simple two-step sonication-centrifugation process, and does not require cell homogenizers, lengthy dialysis steps or run-off reaction. It is simple to execute in a short period of time and does not require high level of laboratory expertise. Thus, it can help facilitate cell-free expression as a standard for translational academic research and industrial process design.
This protocol expands the toolkit available for investigation and utility of V. natriegens, a non-model organism with unique biological properties. Higher protein yield can be achieved by employing semi- or fully-continuous cell-free reactions, to allow for energy regeneration, resupplying of amino acids, and the removal of waste products3,5,27. Furthermore, the engineering of wild-type V. natriegens to produce DNAse- or RNAse-deficient strains, removal of deleterious and competing metabolic pathways, and expression of additional tRNAs could greatly enhance the production of proteins in this system28,29. As we unravel the biology underlying its rapid growth, further development of V. natriegens cell-free systems may accelerate bioproduction capabilities and enable robust expression of therapeutic peptides, small molecules, and synthetic materials.

Cell-free protein synthesis is a versatile and cost-effective method for the expression of valuable proteins or peptides1,2,3,4. Historically, cell-free protein synthesis has been performed using Escherichia coli expression systems; however, there has been a recent surge in using alternative, non-model organisms with novel properties as chassis for cell-free expression5,6,7,8,9,10,11,12. Organisms with unique metabolic profiles are prime candidates as alternatives to E. coli cell-free systems. For example, the marine bacterium Vibrio natriegens is the fastest growing of all known organisms with an observed doubling time of less than 10 min13. This has garnered V. natriegens considerable attention as an emerging microbial host for research and biotechnology14,15,16,17,18. Given that the rapid growth rate of V. natriegens has been linked to high rates of protein synthesis and metabolic efficiency19,20,21, harnessing its cellular machinery for cell-free synthesis may significantly expand the toolkit for rapid protein production and high-throughput screening.
Recently, a cell-free V. natriegens expression system has been demonstrated which is capable of producing super folder GFP (sfGFP) at concentrations of &#62; 260 &#956;g/mL in 3 h with a T7 promoter9. The overall aim of developing this method was to provide users with a highly accessible, cost-efficient, reproducible, and high-yielding cell-free protein expression system that can be prepared using common lab equipment in a short amount of time. This protocol utilizes 1 L cultures in shake flasks, cell lysis by pulse sonication, and small-scale batch reactions in a 96- or 384-well format to maximize parallelization and screening throughput. A long, sustained protein expression is made possible by supplementation of 3-phosphoglyceric acid (3-PGA) as an energy source8,22,23. Upon successfully completing this protocol, a user will have the capability to express a desired protein or set of proteins in a cell-free format using V. natriegens crude cell extract.
Starting from a glycerol stock, V. natriegens crude cell extracts are prepared from cells harvested at an optical density at 600 nm (OD600) of 1.0. A 1 L culture will yield approximately 2&#8722;3 mL of extract, which is sufficient for more than 800 cell-free reactions at 25% crude cell extract. Proteins can be expressed using plasmid DNA, linear DNA, or mRNA template; however, linear DNA template degradation by endogenous nucleases remains a major drawback when using wild type V. natriegens cell-free system9. Starting from V. natriegens cultures, protein useable for downstream applications can be achieved by a single user in 1&#8722;2 full days.

<table><tbody><tr><td>15 mL Tubes</td><td>Corning</td><td>352196</td><td></td></tr><tr><td>2 mL Tubes</td><td>Eppendorf</td><td>22363352</td><td></td></tr><tr><td>384-well Black Assay Plates</td><td>Corning</td><td>3544</td><td></td></tr><tr><td>384-well PCR Plates</td><td>Eppendorf</td><td>951020702</td><td></td></tr><tr><td>50 mL Tubes</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>96-well PCR Plates</td><td>Eppendorf</td><td>30129300</td><td></td></tr><tr><td>Adenosine 3&#039;,5&#039;-cyclic monophosphate sodium salt monohydrate</td><td>Sigma</td><td>A6885</td><td></td></tr><tr><td>Adenosine 5&#039;-triphosphate - 100 mM</td><td>NEB</td><td>N0450L</td><td></td></tr><tr><td>Applied Biosystems Veriti 384-well Thermocycler</td><td>ThermoFisher</td><td>4388444</td><td></td></tr><tr><td>Assay Plate Adhesives</td><td>BioRad</td><td>MSB1001</td><td></td></tr><tr><td>&amp;beta;-Nicotinamide adenine dinucleotide hydrate (NAD)</td><td>Sigma/Roche</td><td>10127965001</td><td></td></tr><tr><td>Coenzyme A hydrate</td><td>Sigma</td><td>C4283</td><td></td></tr><tr><td>Cytidine 5&#039;-triphosphate - 100 mM</td><td>NEB</td><td>N0450L</td><td></td></tr><tr><td>D-(-)-3-Phosphoglyceric acid disodium salt (3-PGA)</td><td>Sigma</td><td>P8877</td><td></td></tr><tr><td>Dewar Flask - 4L</td><td>ThermoScientific</td><td>10-194-100C</td><td></td></tr><tr><td>DL-Dithiothreitol solution - 1 M</td><td>Sigma</td><td>42816</td><td></td></tr><tr><td>Folinic acid calcium salt hydrate</td><td>Sigma</td><td>47612</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Sigma</td><td>A6283</td><td></td></tr><tr><td>Guanosine 5&#039;-triphosphate - 100 mM</td><td>NEB</td><td>N0450L</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>L-Glutamic acid hemimagnesium salt tetrahydrate</td><td>Sigma</td><td>49605</td><td></td></tr><tr><td>L-Glutamic acid potassium salt monohydrate</td><td>Sigma</td><td>G1149</td><td></td></tr><tr><td>LB Broth (Miller)</td><td>Sigma</td><td>L3522</td><td></td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>Plasmid pJL1-sfGFP</td><td>Addgene</td><td>69496</td><td></td></tr><tr><td>Plasmid Plus &lt;em&gt;Maxi&lt;/em&gt; kit</td><td>Qiagen</td><td>12963</td><td></td></tr><tr><td>Poly(ethylene glycol) (PEG)-8000</td><td>Sigma</td><td>89510</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>Potassium hydroxide Pellets</td><td>Sigma/Roche</td><td>1050121000</td><td></td></tr><tr><td>Q125 Sonicator and CL-18 probe with a ?-inch tip</td><td>Qsonica</td><td>4422</td><td></td></tr><tr><td>RNA Clean and Concentrator Kit</td><td>Zymo</td><td>R1013</td><td></td></tr><tr><td>RNase Inhibitor, Murine</td><td>NEB</td><td>M0314</td><td></td></tr><tr><td>RTS Amino Acid Sampler</td><td>Biotechrabbit</td><td>BR1401801</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Spermidine</td><td>Sigma</td><td>S0266</td><td></td></tr><tr><td>T7 RNA Polymerase</td><td>NEB</td><td>M0251</td><td></td></tr><tr><td>Tris Solution (pH 8.0) - 1 M</td><td>Invitrogen</td><td>AM9856</td><td></td></tr><tr><td>tRNA from E. coli MRE 600</td><td>Sigma/Roche</td><td>10109541001</td><td></td></tr><tr><td>Uridine 5&#039;-triphosphate - 100 mM</td><td>NEB</td><td>N0450L</td><td></td></tr><tr><td>&lt;em&gt;Vibrio natriegens &lt;/em&gt;(Wild-type) Lyophilized Stock</td><td>ATCC</td><td>14048</td><td></td></tr></tbody></table>

cell-free protein expression using the rapidly growing bacterium vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

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Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...

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Research

JoVE Journal

Bioengineering

17.3K Views.  Harvard Medical School. Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

Video: Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene4. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)5 (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism6 and other representative of the genus Vibrio such as V. fischeri7. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region8. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.
This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences5. Several optimization steps concerning the initial protocol of chitin-induced natural transformation6 are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes8, the donation of PCR-derived DNA as transforming material9, and the addition of FLP-recombination target sites (FRT)5. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase10.

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

Immunology and Infection

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

Biochemistry

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

CFPS Protocols for Micro-Compartmentalized Synthetic Cells

Cell-Free Protein Synthesis System for Building Synthetic Cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

Author Spotlight: Optimizing CFPS Systems for Synthetic Cell Construction

This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell development.

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Biology

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous ...

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. 
This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (&lt;1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. 
Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

This protocol describes using commercial, cell-free protein expression kits to produce membrane proteins supported in nanodisc that can be used as antigens in subunit vaccines.

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard ...

Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate

Cell-free gene expression offers the power of biology without the complications of a living organism. Although many such gene expression systems exist, most are quite expensive to buy and/or require special equipment and finely honed expertise to produce effectively. This protocol describes a method to produce bacterial cell-free lysate that supports high levels of gene expression, using only standard laboratory equipment and requiring minimal processing. The method uses an Escherichia coli strain producing an endolysin that does not affect growth, but which efficiently lyses a harvested cell pellet following a simple freeze-thaw cycle. The only further processing required is a brief incubation followed by centrifugation to clear the autolysate of cellular debris. Dynamic gene circuits can be achieved through heterologous expression of the ClpX protease in the cells before harvesting. An E. coli strain lacking the lacZ gene can be used for high-sensitivity, cell-free biosensing applications using a colorimetric or fluorescent readout. The entire protocol requires as few as 8-9 hours, with only 1-2 hours of hands-on labor from inoculation to completion. By reducing the cost and time to obtain cell-free lysate, this method should increase the affordability of cell-free gene expression for various applications.

This protocol describes a rapid and simple method to produce bacterial lysate for cell-free gene expression, using an engineered strain of Escherichia coli and requiring only standard laboratory equipment.

Cell-free gene expression offers the power of biology without the complications of a living organism. Although many such gene expression systems exist, ...

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

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TransFLP — A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

Cell-Free Protein Synthesis System for Building Synthetic Cells

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate