title: Cell-Free Protein Synthesis in Miniaturized Array Devices and Effects of Device Orientation.
abstract: Cell-free protein synthesis (CFPS) has been used as an alternative to cell-based recombinant technology for protein production in academic and industrial labs. The continuous-exchange format generally has higher expression yield by constantly supplying a nutrient solution and removing inhibitory by-products through a porous membrane. Because of the concern of possible membrane clogging by large molecules in the CFPS solution, we investigated the effects of membrane orientation on protein synthesis. We fabricated a miniaturized array device called Vertical-I with its membrane oriented vertically in reference to the table surface and found that the protein synthesis yield in the Vertical-I device was 144% higher than the Horizontal Device reported previously. The reaction time was also faster; β-glucuronidase reached the synthesis yield plateau after 2 h in the Vertical-I device versus 4 h in the Horizontal Device. Possible clogging of membrane pores was confirmed by fluorescein diffusion measurement. Using these results, we designed a device called Vertical-II that would fit into a 96 well plate holder for compatibility with commercial reagent dispensers and microplate readers. The experimentally optimized device increased protein expression 406% over the Horizontal Device and consumes 5 times fewer reagents than a commercial device, showing the potential for high-throughput protein synthesis.
authors.affiliation: J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Jackson
authors.affiliation: J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA Interdisciplinary Microsystems Group, Department of Mechanical and Aerospace Engineering, University of Florida, Gainesville, FL, USA Department of Chemistry, University of Florida, Gainesville, FL, USA hfan@ufl.edu.
authors.forename: Z Hugh
authors.initials: ZH
authors.lastname: Fan
mesh_terms: D019149:Bioreactors; D002474:Cell-Free System / Q000378:metabolism*; D004867:Equipment Design; D006874:Hydrostatic Pressure; D008567:Membranes, Artificial; D008904:Miniaturization / Q000295:instrumentation*; D011994:Recombinant Proteins / Q000378:metabolism*; D014908:Triticum / Q000378:metabolism
chemical_list: D008567:Membranes, Artificial; D011994:Recombinant Proteins
keywords: array; cell free; microreactors; protein synthesis; vertical orientation