Advantages of Cell-Free Protein Expression

Cell-free protein expression offers several advantages over traditional methods for protein production. We discuss those here and look at how you can bring cell-free protein expression into your lab.

Traditional protein expression methods present multiple challenges

Traditional methods for producing recombinant proteins begin with introducing a DNA template for target expression into a host cell line. E. coli cell lines are most commonly used, but yeast, insect, and mammalian cell lines are also popular for the greater protein complexity they can provide. Once the cells have been expanded in culture, the expressed protein is recovered directly from the media (secreted proteins) or extracted following cell lysis. “Often, the protein will have an affinity tag to simplify purification,” reports Sreethu Sankar, Product Manager at Proteintech. “However this has the potential to interfere with the active site, resulting in an inactive product.”

Penny Jensen, Ph.D., Staff Scientist at Thermo Fisher Scientific, notes that limitations of traditional methods include low or no expression, misfolded and therefore non-functional proteins, and solubility issues leading to proteins being expressed only in inclusion bodies. Solubility can be especially problematic when expressing membrane proteins, which have large numbers of hydrophobic amino acids. “Other challenges that must be overcome include the presence of contaminating host proteases, which can lead to target degradation and levels of endotoxin,” she explains. “In addition, there is the requirement for a cell culture facility with the expertise to maintain, grow, and harvest cells.”

Unfortunately, the challenges don’t stop there. Robin Hurst, Senior Research Scientist at Promega, comments that because cells can be sensitive to toxic proteins, expression of targets such as DNA nucleases can be dismal. “Also, cells don’t allow for easy optimization,” she says. “Optimization is often needed for recombinant proteins that are difficult to express, or if labeled or unnatural amino acids are warranted.” Scale up presents further problems, particularly when working with mammalian cell lines. For these reasons, cell-free protein expression may often represent a better option.

Cell-free protein expression addresses common problems

Cell-free protein expression uses cellular lysates instead of living cells as a source of components required for protein synthesis. “With cell-free protein expression, you simply add an mRNA, DNA, or PCR template to the chosen expression system and incubate for a designated time,” says Hurst. “The only DNA or PCR template requirement is that the DNA must have the correct promoter for the system —T7, SP6, or T3.”

According to James C. Samuelson, Ph.D., Senior Scientist within the Protein Expression and Modification Division at New England Biolabs, the template typically encodes a phage T7 promoter and T7 terminator sequence. “Accordingly, DNA-templated reactions are supplemented with purified T7 RNA polymerase to enable one-pot transcription and translation of the target,” he says.

Samuelson also explains that cell-free protein expression is most commonly performed using either an E. coli cell lysate (S30 extract) or a fully reconstituted system based upon E. coli translation machinery. “Reconstitution of E. coli protein synthesis in a test tube may be accomplished by purification and formulation of 35 proteins along with ribosomes and tRNAs. Other supplements are also required, such as those necessary for energy regeneration, to achieve sustained target protein expression.”

Advantages of cell-free protein expression

“Advantages of cell-free protein expression include its ease of use, rapid protein production, and minimal requirements for lab space, equipment, and expertise compared to traditional methods,” says Jensen. “Plus, cell-free protein expression lets researchers incorporate unnatural labels or amino acids into targets of interest, as well as express toxic proteins.” These features of cell-free protein expression have seen its uptake for performing functional assays, investigating protein interactions, and screening translational inhibitors, as well as for ribosomal displays.

“Another important advantage of cell-free protein expression is that it is easier to optimize,” adds Hurst. “For example, researchers can readily assess the impact of adding folding enzymes, amber suppressor amino acid machinery, amino acids with stable isotopes, or pre-charged tRNAs. The set up and downstream applications are also more straightforward with cell-free systems. Not only are there no transformations, but there is no need for lysis or a subsequent spin during purification or co-immunoprecipitations.”

Samuelson points out that the use of linear DNA templates enables high-throughput workflows where researchers can proceed from amplification of synthetic DNA to the expression of target proteins in a matter of hours. “Synthetic linear DNA also represents a practical choice due to its low cost, convenience, and the ability to rapidly amplify it by PCR or rolling circle amplification,” he says. “Additionally, cell-free protein expression with linear DNA or mRNA templates eliminates any concern of plasmid genetic instability that may occur during traditional E. coli based expression.”

Lastly, Sankar notes that bacterial cell-free protein expression systems can help labs save a significant amount of money. “Recently, it has been shown that introducing novel energy sources into cell-free protein expression processes can dramatically increase protein yields, thus reducing the cost of production,” he says. It is perhaps therefore unsurprising that cell-free protein expression is receiving a great deal of interest from the wider scientific community.

Supporting cell-free protein expression

When it comes to introducing cell-free protein expression into your lab, many different products and services are available. “New England Biolabs offers two cell-free protein expression systems—the NEBExpress® Cell-free E. coli Protein Synthesis System, which is extract-based, and the PURExpress® In Vitro Protein Synthesis Kit, which is a reconstituted protein synthesis system,” says Samuelson. “Both of these enable protein synthesis in just a few hours and, in most cases, allow target protein function to be analyzed directly.”

Thermo Fisher Scientific offers cell-free protein expression systems derived from E. coli, rabbit reticulocyte, and mammalian cells. “The main advantages of using the mammalian system are that it allows for expression of functional, high molecular weight proteins, with the incorporation of post-translational modifications,” notes Jensen. “Two different mammalian kits are available depending on the amount of protein required—the 1-Step Human Coupled Kit and a dialysis-based 1-Step Human High Yield.”

Promega’s cell-free protein expression products include multiple prokaryotic and eukaryotic cell-free systems. “Our cornerstone product in this area is the mammalian-based TNT^® Quick-Coupled Transcription/Translation System, which is available in both T7 and SP6 versions,” reports Hurst. “The PCR template version, TNT^® T7 Quick for PCR DNA, can be used to screen PCR reactions, while mRNA template versions allow for translation to be optimized. We also offer prokaryotic systems for high yield protein expression (500 μg/mL), as well as products for tagging expressed proteins.”