Abstract
Recombinant protein expression and purification is an essential component of biomedical research and drug discovery. Advances in automation and laboratory robotics have enabled the development of highly parallel and rapid processes for cell culture and protein expression, purification, and analysis. Human embryonic kidney (HEK) cells and Chinese hamster ovary (CHO) cells have emerged as the standard host cell workhorses for producing recombinant secreted mammalian proteins by using both transient and stable production strategies. In this chapter we describe a fully automated custom platform, Protein Expression and Purification Platform (PEPP), used for transient protein production from HEK cells and stable protein production from CHO cells. Central to PEPP operation is a suite of custom robotic and instrumentation platforms designed and built at GNF, custom cell culture ware, and custom scheduling software referred to as Runtime. The PEPP platform enables cost-effective, facile, consistent production of proteins at quantities and quality useful for early stage drug discovery tasks such as screening, bioassays, protein engineering, and analytics.
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References
Geisse S (2009) Reflections on more than 10 years of TGE approaches. Protein Expr Purif 64(2):99–107. https://doi.org/10.1016/j.pep.2008.10.017
Kim JY, Kim YG, Lee GM (2012) CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol 93(3):917–930. https://doi.org/10.1007/s00253-011-3758-5
Dalton AC, Barton WA (2014) Over-expression of secreted proteins from mammalian cell lines. Protein Sci 23(5):517–525. https://doi.org/10.1002/pro.2439
Suen KF, Turner MS, Gao F, Liu B, Althage A, Slavin A, Ou W, Zuo E, Eckart M, Ogawa T, Yamada M, Tuntland T, Harris JL, Trauger JW (2010) Transient expression of an IL-23R extracellular domain Fc fusion protein in CHO vs. HEK cells results in improved plasma exposure. Protein Expr Purif 71(1):96–102. https://doi.org/10.1016/j.pep.2009.12.015
Croset A, Delafosse L, Gaudry JP, Arod C, Glez L, Losberger C, Begue D, Krstanovic A, Robert F, Vilbois F, Chevalet L, Antonsson B (2012) Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells. J Biotechnol 161(3):336–348. https://doi.org/10.1016/j.jbiotec.2012.06.038
Bandaranayake AD, Almo SC (2014) Recent advances in mammalian protein production. FEBS Lett 588(2):253–260. https://doi.org/10.1016/j.febslet.2013.11.035
Bos AB, Luan P, Duque JN, Reilly D, Harms PD, Wong AW (2015) Optimization and automation of an end-to-end high throughput microscale transient protein production process. Biotechnol Bioeng 112(9):1832–1842. https://doi.org/10.1002/bit.25601
Gonzalez R, Jennings LL, Knuth M, Orth AP, Klock HE, Ou W, Feuerhelm J, Hull MV, Koesema E, Wang Y, Zhang J, Wu C, Cho CY, Su AI, Batalov S, Chen H, Johnson K, Laffitte B, Nguyen DG, Snyder EY, Schultz PG, Harris JL, Lesley SA (2010) Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency. Proc Natl Acad Sci U S A 107(8):3552–3557. https://doi.org/10.1073/pnas.0914019107
Rue SM, Anderson PW, Miller JM, Fanale SG, Chang JY, Glaser SM, Lesley SA (2018) Mammalian cell culture density determination using a laser through-beam sensor. BioTechniques 65(4):224–226. https://doi.org/10.2144/btn-2018-0059
Plasmid DNA Purification User Manual (2015) Macherey-Nagel. http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NS96.pdf. Accessed 22 Mar 2017
Application Notes-Tecan (2017) Macherey-Nagel. http://www.mn-net.com/tabid/12425/default.aspx. Accessed 11 Apr 2017
FreeStyle 293 Expression System (2010) Invitrogen. https://tools.thermofisher.com/content/sfs/manuals/freestyle293_system_man.pdf. Accessed 11 Mar 2017
TR0006.4 Extinction Coefficients (2013) Thermo Fisher Scientific. https://tools.thermofisher.com/content/sfs/brochures/TR0006-Extinction-coefficients.pdf. Accessed 10 Mar 2017
E-Gel Precast Agarose Gels. Thermo Fisher Scientific. https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/nucleic-acid-gel-electrophoresis/e-gel-electrophoresis-system/e-gel-pre-cast-agarose-gels.html. Accessed 22 Mar 2017
Nucleofector Protocols (2012) Lonza. http://bio.lonza.com/resources/product-instructions/protocols. Accessed 22 Mar 2017
Amaxa™ 4D-Nucleofector™ Optimization Protocol for Cell Lines For 4D-Nucleofector™ X Unit–Transfection in suspension (2010) Lonza. http://www.lonzabio.jp/catalog/pdf/ri/I548.pdf. Accessed 13 Mar 2017
Acknowledgements
The authors would like to acknowledge Salvatore Fanale, Analisa Benedetto, Heath Klock, Julie Vance, Melisa Low, Sarah Cox, Daniel McMullan, Mark Knuth, Marc Gustafson, Jim Chang, Marie Smith, Daniel Sipes, and James Mainquist for their contributions to the protocols and technologies described in this chapter.
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Rue, S.M., Anderson, P.W., Gaylord, M.R., Miller, J.J., Glaser, S.M., Lesley, S.A. (2019). A High-Throughput System for Transient and Stable Protein Production in Mammalian Cells. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9624-7_5
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DOI: https://doi.org/10.1007/978-1-4939-9624-7_5
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