A Brief History of Extract-Based Cell-Free Protein Expression Systems

by Aidan Tinafar January 4, 2022

Cell-free protein expression has exploded in popularity in recent years. The ability to go from DNA templates to purified proteins in hours, rather than days or weeks, has the potential to revolutionize synthetic biology as an industry. One common way to prepare cell-free protein expression systems is to use the translation (and sometimes transcription) machinery obtained from crude cell extracts. To fully appreciate the excitement surrounding extract-based cell-free systems, it is worth taking a look at their emergence and evolution from a primarily research-based tool for furthering our understanding of how biological systems work to their use in biotechnological applications.

Origins

The first ever cell-free protein expression system was published in 1954 by Joan Folkes and Ernest Gale, who were both subsequently nominated for the Nobel Prize in chemistry in 1956. In their seminal paper, they write:

It has frequently been suggested in recent literature that nucleic acids have an organizing or controlling role in protein synthesis.

…

But any proof of the part they play, let alone its mechanism, awaits the preparation of an experimental system which can synthesize protein and on which an effect of nucleic acids can be demonstrated. Such a system has been obtained by disruption suspensions of Staphylococcus aureus by exposure to supersonic vibration.

The first E. coli-based system appears to have been described by Marvin Lamborg and Paul Zamecnik in 1960. Soon thereafter, in 1961, Heinrich Matthaei and Marshall Nirenberg published their version of an E. coli-based system, which was subsequently used for deciphering the genetic code and earning Nirenberg (among others) the 1968 Nobel Prize in Medicine. Despite previous publications indicating the contrary, the initial discovery of extract-based cell-free systems has, whether implicitly or explicitly, been mistakenly attributed by some to Nirenberg and Matthaei. It is perhaps of note that one of the two original inventors, namely Joan Folkes, was a young woman – a fact which may have contributed to this important historical erasure.

A detailed protocol published in 1973 by Geoffrey Zubay, which makes “minor modifications” to the Nirenberg and Matthaei method, forms the basis of the widely used extract-based E. coli systems that we know today. This protocol calls for lysate clarification steps requiring centrifugation at 30,000 g; accordingly, many refer to the resulting lysate as the S30 extract. It was later shown that centrifugation at 12,000 g was sufficient for clarifying the lysate from many E. coli strains. However, despite initial efforts to coin the term S12 for extracts made by such modification, the term S12 (and its counterparts such as S18) never really took hold and many continue to use the term S30 regardless of the applied centrifugal force.

Evolution of the PANOx-SP system

Perhaps the most popular cell-free system used by many labs today is the PANOx-SP system. PANOx stands for phosphoenolpyruvate (PEP), amino acids, NAD+ and oxalic acid. The initial PANOx system was developed by Dong-Myung Kim and James Swartz in 2001 as a method of prolonging cell-free reactions by enabling regeneration of adenosine triphosphate (ATP) from a conventional energy sources (i.e. PEP) without the need for exogenous enzymes.

In 1979, Jelenc and Kurland reported that naturally occurring polyamines (i.e. spermidine and putrescine) can improve “the extent and fidelity of translation”. Relying on this discovery, a series of optimizations experiments were carried by Michael Jewett and James Swartz to investigate the effects of substituting polyethylene glycol (PEG) [the use of which can, for example, be traced back to the 1973 Zubay protocol] with spermidine and putrescine. In their article, published in 2004, they coined their newly designed system PANOx-SP.

It is important to note that the PANOx-SP system is only one of the many great designs used by labs around the globe today. Moreover, cell-free expression systems have been prepared using extracts from a wide range of organisms including bacteria, protozoans, plants, insects, mammals and fungi. The optimal formulation and source of the extract will ultimately depend on the specific application. Modern high yield-systems such as the PANOx-SP formulations using E. coli-based extracts have transformed extract-based cell-free systems from being tools that further our understanding of biological systems to powerful platforms for synthetic biology applications.

A Note on the Future of Cell-Free Protein Expression Systems

Attainable yield from cell-free protein expression reactions has now reached a point where comparable amounts of protein can be obtained whether cells are grown while expressing a target protein or are grown without a template, converted into an extract and then used to express a target. This is not a trivial improvement. It means that we can now separate the task of fermentation from protein production.

Proteins tend to be expensive at small scale, and relatively cheap at scale. For individual labs, having to grow a culture for each small-batch expression is not cost-effective. Since cell-free systems separate protein expression from fermentation, economies of scale can be achieved by carrying out large cultures at low marginal costs and providing the resulting production capacity to labs via extract production. This means that more labs will be able to carry out small-scale protein expression at low cost. With the rapidly declining cost of DNA synthesis, it is likely that cell-free protein expression systems will usher in a new era of phenotyping that was previously unimaginable.

Recognizing the potential for cell-free systems in accelerating life sciences, we are building the tools to enable small-scale custom protein production by research labs, without the need for specialized equipment. Learn more about our high-yield cell-free protein expression reagents and bioreactors on our website.

Special thanks to Alexander Klenov, Taylor Sheahan and Keith Pardee for their edits and significant contributions to this post.