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Optimizing cell‐free protein expression in CHO: Assessing small molecule mass transfer effects in various reactor configurations
Author(s) -
PeñalberJohnstone Chariz,
Ge Xudong,
Tran Kevin,
Selock Nicholas,
Sardesai Neha,
Gurramkonda Chandrasekhar,
Pilli Manohar,
Tolosa Michael,
Tolosa Leah,
Kostov Yordan,
Frey Douglas D.,
Rao Govind
Publication year - 2017
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26282
Subject(s) - chinese hamster ovary cell , chemistry , cell free protein synthesis , bioreactor , chromatography , transfer rna , biochemistry , creatine , phosphate , bioprocess , biophysics , protein biosynthesis , biology , rna , organic chemistry , receptor , paleontology , gene
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.

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