Protein therapeutics are powerful tools in the fight against diabetes, cancers, growth disorders, and many other debilitating diseases. However, availability is limited due to cost and complications of production from living organisms. To make life‐saving protein therapeutics more available to the world, the possibility of magistral or point‐of‐care protein therapeutic production has gained focus. The recent invention and optimization of lyophilized “cell‐free” protein synthesis reagents and its demonstrated ability to produce highly active versions of FDA‐approved cancer therapeutics have increased its potential for low‐cost, single‐batch, magistral medicine. Here we present for the first time the concept of increased oxygen mass transfer in small‐batch, cell‐free protein synthesis (CFPS) reactions through air‐water foams. These “hydrofoam” reactions increased CFPS yields by up to 100%. Contrary to traditional protein synthesis using living organisms, where foam bubbles cause cell‐lysis and production losses, hydrofoam CFPS reactions are “cell‐free” and better tolerate foaming. Simulation and experimental results suggest that oxygen transfer is limiting in even small volume batch CFPS reactors and that the hydrofoam format improved oxygen transfer. This is further supported by CFPS reactions achieving higher yields when oxygen gas replaces air in the headspace of batch reactions. Improving CFPS yields with hydrofoam reduces the overall cost of biotherapeutic production, increasing availability to the developing world. Beyond protein therapeutic production, hydrofoam CFPS could also be used to enhance other CFPS applications including biosensing, biomanufacturing, and biocatalysis.
Cell‐free protein synthesis (CFPS) is a versatile biotechnology platform enabling a broad range of applications including clinical diagnostics, large‐scale production of officinal therapeutics, small‐scale on‐demand production of personal magistral therapeutics, and exploratory research. The shelf stability and scalability of CFPS systems also have the potential to overcome cost and infrastructure challenges for distributing and using essential medical tests at home in both high‐ and low‐income countries. However, CFPS systems are often more time‐consuming and expensive to prepare than traditional in vivo systems, limiting their broader use. Much work has been done to lower CFPS costs by optimizing cell extract preparation, small molecule reagent recipes, and DNA template preparation. In order to further reduce reagent cost and preparation time, this work presents a CFPS system that does not require separately purified DNA template. Instead, a DNA plasmid encoding the recombinant protein is transformed into the cells used to make the extract, and the extract preparation process is modified to allow enough DNA to withstand homogenization‐induced shearing. The finished extract contains sufficient levels of intact DNA plasmid for the CFPS system to operate. For a 10 mL scale CFPS system expressing recombinant sfGFP protein for a biosensor, this new system reduces reagent cost by more than half. This system is applied to a proof‐of‐concept glutamine sensor compatible with smartphone quantification to demonstrate its viability for further cost reduction and use in low‐resource settings.
more » « less- PAR ID:
- 10442966
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology Progress
- Volume:
- 39
- Issue:
- 3
- ISSN:
- 8756-7938
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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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.more » « less
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