title: Optimizing cell-free protein expression in CHO: Assessing small molecule mass transfer effects in various reactor configurations.
abstract: 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.
authors.affiliation: Center for Advanced Sensor Technology, University of Maryland Baltimore County, Baltimore County, 1000 Hilltop Circle, TRC 252, Baltimore, Maryland 21250.
authors.forename: Chariz
authors.initials: C
authors.lastname: Peñalber-Johnstone
authors.forename: Xudong
authors.initials: X
authors.lastname: Ge
authors.affiliation: Center for Advanced Sensor Technology, University of Maryland Baltimore County, Baltimore County, 1000 Hilltop Circle, TRC 252, Baltimore, Maryland 21250.
authors.forename: Kevin
authors.initials: K
authors.lastname: Tran
authors.affiliation: Center for Advanced Sensor Technology, University of Maryland Baltimore County, Baltimore County, 1000 Hilltop Circle, TRC 252, Baltimore, Maryland 21250.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Selock
authors.affiliation: Center for Advanced Sensor Technology, University of Maryland Baltimore County, Baltimore County, 1000 Hilltop Circle, TRC 252, Baltimore, Maryland 21250.
authors.forename: Neha
authors.initials: N
authors.lastname: Sardesai
authors.affiliation: Center for Advanced Sensor Technology, University of Maryland Baltimore County, Baltimore County, 1000 Hilltop Circle, TRC 252, Baltimore, Maryland 21250.
authors.forename: Chandrasekhar
authors.initials: C
authors.lastname: Gurramkonda
authors.affiliation: Center for Advanced Sensor Technology, University of Maryland Baltimore County, Baltimore County, 1000 Hilltop Circle, TRC 252, Baltimore, Maryland 21250.
authors.forename: Manohar
authors.initials: M
authors.lastname: Pilli
authors.affiliation: Center for Advanced Sensor Technology, University of Maryland Baltimore County, Baltimore County, 1000 Hilltop Circle, TRC 252, Baltimore, Maryland 21250.
authors.forename: Michael
authors.initials: M
authors.lastname: Tolosa
authors.affiliation: Center for Advanced Sensor Technology, University of Maryland Baltimore County, Baltimore County, 1000 Hilltop Circle, TRC 252, Baltimore, Maryland 21250.
authors.forename: Leah
authors.initials: L
authors.lastname: Tolosa
authors.affiliation: Center for Advanced Sensor Technology, University of Maryland Baltimore County, Baltimore County, 1000 Hilltop Circle, TRC 252, Baltimore, Maryland 21250.
authors.forename: Yordan
authors.initials: Y
authors.lastname: Kostov
authors.affiliation: Center for Advanced Sensor Technology, University of Maryland Baltimore County, Baltimore County, 1000 Hilltop Circle, TRC 252, Baltimore, Maryland 21250.
authors.forename: Douglas D
authors.initials: DD
authors.lastname: Frey
authors.affiliation: Center for Advanced Sensor Technology, University of Maryland Baltimore County, Baltimore County, 1000 Hilltop Circle, TRC 252, Baltimore, Maryland 21250.
authors.forename: Govind
authors.identifier: 0000-0001-6140-7582
authors.initials: G
authors.lastname: Rao
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000737:chemistry / Q000378:metabolism*; D002474:Cell-Free System / Q000378:metabolism*; D003412:Cricetulus; D014176:Protein Biosynthesis / Q000502:physiology*; D015202:Protein Engineering / Q000295:instrumentation* / Q000379:methods*; D011786:Quality Control
keywords: Chinese hamster ovary cells; bioreactors; cell-free protein synthesis; continuous-exchange cell-free; green fluorescent protein; in vitro protein expression