Cell-Free Protein Synthesis in Miniaturized Array Devices and Effects of Device Orientation

Thanks to the synthetic biology, the laborious and restrictive procedure for producing a target protein in living microorganisms by biotechnological approaches can now experience a robust, pliant yet efficient alternative. The new system combined with lab‐on‐chip microfluidic devices and nanotechnology offers a tremendous potential envisioning novel cell‐free formats such as DNA brushes, hydrogels, vesicular particles, droplets, as well as solid surfaces. Acting as robust microreactors/microcompartments/minimal cells, the new platforms can be tuned to perform various tasks in a parallel and integrated manner encompassing gene expression, protein synthesis, purification, detection, and finally enabling cell‐cell signaling to bring a collective cell behavior, such as directing differentiation process, characteristics of higher order entities, and beyond. In this review, we issue an update on recent cell‐free protein synthesis (CFPS) formats. Furthermore, the latest advances and applications of CFPS for synthetic biology and biotechnology are highlighted. In the end, contemporary challenges and future opportunities of CFPS systems are discussed.

Section: Challenges and Future Directionsmentioning

confidence: 99%

Section: Challenges and Future Directionsmentioning

confidence: 99%

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Ayoubi‐Joshaghani

Dianat‐Moghadam

Seidi

et al. 2020

“…A consensus exists in the published data highlighting that among the various ways in which to carry out the expression, optimum yields are obtained using a continuous‐exchange cell‐free (CECF) format (Jackson et al, ; Schwarz et al, ). Several efforts in the development of automated and high‐throughput cell‐free systems have been done (Aoki et al, ) including recent significant advances in bioreactor configurations for small‐scale protein production (Jackson and Fan, ; Jackson et al, ; Millet et al, ; Timm et al, , ). Along with the crucial endeavor of optimizing the CFPS system by fine‐tuning the reaction components and tweaking the biological machinery (Brödel et al, ; Schoborg et al, ), progress in the equally vital work of designing the most efficient bioreactor is indispensable.…”

Section: Introductionmentioning

confidence: 99%

Optimizing cell‐free protein expression in CHO: Assessing small molecule mass transfer effects in various reactor configurations

Peñalber-Johnstone

Andar

Tran

et al. 2017

Cell-free protein synthesis (CFPS) is an ideal platform for rapid and convenient protein production. However, bioreactor design remains a critical consideration in optimizing protein expression. Using turbo green fluorescent protein (tGFP) as a model, we tracked small molecule components in a Chinese Hamster Ovary (CHO) CFPS system to optimize protein production. Here, three bioreactors in continuous-exchange cell-free (CECF) format were characterized. A GFP optical sensor was built to monitor the product in real-time. Mass transfer of important substrate and by-product components such as nucleoside triphosphates (NTPs), creatine, and inorganic phosphate (Pi) across a 10-kDa MWCO cellulose membrane was calculated. The highest efficiency measured by tGFP yields were found in a microdialysis device configuration; while a negative effect on yield was observed due to limited mass transfer of NTPs in a dialysis cup configuration. In 24-well plate high-throughput CECF format, addition of up to 40 mM creatine phosphate in the system increased yields by up to ∼60% relative to controls. Direct ATP addition, as opposed to creatine phosphate addition, negatively affected the expression. Pi addition of up to 30 mM to the expression significantly reduced yields by over ∼40% relative to controls. Overall, data presented in this report serves as a valuable reference to optimize the CHO CFPS system for next-generation bioprocessing. Biotechnol. Bioeng. 2017;114: 1478-1486. © 2017 Wiley Periodicals, Inc.

“…To achieve high‐throughput protein expression and reduce reagent volumes and cost, CFPS is often performed in miniaturized or microfluidic devices (Angenendt et al, ; Agresti et al, ; Biyani et al, ; Fallah‐Araghi et al, ; Hahn et al, ; Jackson et al, ; Jackson and Fan, ; Khnouf et al, ; Khnouf et al, ; Mei et al, ; Okano et al, ; Osaki et al, ; Park et al, ; Teh et al, ; Wu et al, ; Wu et al, ). However, in the CECF format, miniaturization is generally restricted due to the necessity for a porous membrane that allows passive chemical diffusion between reaction and feeding solutions.…”

Section: Introductionmentioning

confidence: 99%

“…However, in the CECF format, miniaturization is generally restricted due to the necessity for a porous membrane that allows passive chemical diffusion between reaction and feeding solutions. In miniaturized systems, the CECF format has been accomplished through the use of microdroplets (Agresti et al, ; Fallah‐Araghi et al, ; Osaki et al, ; Teh et al, ; Wu et al, ; Wu et al, ), a protein‐producing gel (Park et al, ), and microfluidic devices (Hahn et al, ; Jackson and Fan, ; Jackson et al, ; Khnouf et al, ; Mei et al, ). In these microfluidic devices, an array of protein expression microreactors are typically created for the simultaneous expression of numerous proteins with the reaction solution being isolated from the feeding solution by a dialysis or nanoporous membrane (Hahn et al, ; Jackson and Fan, ; Jackson et al, ; Khnouf et al, ; Mei et al, ).…”

Section: Introductionmentioning

confidence: 99%

Optimization of a miniaturized fluid array device for cell‐free protein synthesis

Jackson

Jin

Fan

2015

Self Cite

Cell-free protein synthesis (CFPS), which entails synthesizing proteins outside of intact cells, is conducted in several formats with the continuous-exchange cell-free (CECF) format generally having the greatest protein expression yields. With this format, continuous chemical exchange occurs through a dialysis membrane separating a reaction solution from a feeding solution containing supplemental nutrient/energy molecules. Here, we describe the optimization of the miniaturized fluid array device (µFAD) by studying the effects of structural and experimental parameters responsible for the heightened chemical exchange across the dialysis membranes and enhanced protein expression capabilities of the high-throughput device. The interface area and number between the reaction and feeding solutions have a direct impact on protein expression, with a 1.6% enhancement in protein expression yield with each square millimeter increase in area and a 20% decrease with each additional interface. For nutrient/energy availability, an increasing solution volume ratio and height difference increase protein expression yield until the expression yield plateaus at a volume ratio of 20 to 1 (feeding to reaction solution) and a solution height difference of 2 mm. This yield can be further increased by 7% every 30 min with feeding solution replacement. Of the studied experimental factors (feeding solution stirring, device shaking, and temperature increase), feeding solution stirring has a significant effect on protein expression in this device. In the optimized system, green fluorescent protein (GFP), ß-glucuronidase (GUS), ß-galactosidase (LacZ), luciferase, and tissue plasminogen activator (tPA) expression increased 77.8-, 212-, 3.66-, 463-, and 5.43-fold, respectively, compared to the conventional batch format in a standard microplate. These results highlight the significance of structural/experimental conditions on the productive expression of proteins in the CECF format.