title: Optimization of a miniaturized fluid array device for cell-free protein synthesis.
abstract: 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.
authors.affiliation: J.Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, P.O. Box 116131, Gainesville, 32611, Florida.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Jackson
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, P.O. Box 100266, Gainesville, 32610, Florida.
authors.forename: Shouguang
authors.initials: S
authors.lastname: Jin
authors.affiliation: J.Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, P.O. Box 116131, Gainesville, 32611, Florida. hfan@ufl.edu.|Interdisciplinary Microsystems Group, Department of Mechanical and Aerospace Engineering, University of Florida, P.O. Box 116250, Gainesville, 32611, Florida. hfan@ufl.edu.|Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, 32611, Florida. hfan@ufl.edu.
authors.forename: Z Hugh
authors.initials: ZH
authors.lastname: Fan
mesh_terms: D019149:Bioreactors; D002474:Cell-Free System; D003956:Dialysis; D004867:Equipment Design; D007287:Inorganic Chemicals; D008566:Membranes; D014176:Protein Biosynthesis
chemical_list: D007287:Inorganic Chemicals
keywords: cell-free; high-throughput; microreactors; miniaturized fluid array; protein synthesis