Optimizing Transient Recombinant Protein Expression in Mammalian Cells

  • Ralph F. Hopkins
  • Vanessa E. Wall
  • Dominic EspositoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 801)


Transient gene expression (TGE) in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells. The rapidly increasing need for recombinant proteins requires further improvements in TGE technology. While a great deal of focus has been directed toward optimizing the secretion of antibodies and other naturally secreted targets, much less work has been done on ways to improve cytoplasmic expression in mammalian cells. The benefits to protein production in mammalian cells, particularly for eukaryotic proteins, should be very significant – glycosylation and other posttranslational modifications will likely be native or near-native, solubility and protein folding would likely improve overexpression in heterologous hosts, and expression of proteins in their proper intracellular compartments is much more likely to occur. Improvements in this area have been slow, however, due to limited development of the cell culture processes needed for low-cost, higher-throughput expression in mammalian cells, and the relatively low diversity of DNA vectors for protein production in these systems. Here, we describe how the use of recombinational cloning, coupled with improvements in transfection protocols which increase speed and lower cost, can be combined to make mammalian cells much more amenable for routine recombinant protein expression.

Key words

Optimizing transient expression Gateway cloning Gateway Multisite HEK293E Polyethylenimine 



The authors thank Cammi Bittner, Veronica Roberts, Leslie Garvey, and Kelly Esposito for assistance in protocol development. This work has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Ralph F. Hopkins
    • 1
  • Vanessa E. Wall
    • 2
  • Dominic Esposito
    • 2
    Email author
  1. 1.Protein Expression LaboratorySAIC-Frederick, Inc.FrederickUSA
  2. 2.Protein Expression LaboratorySAIC-Frederick, Inc., National Cancer InstituteFrederickUSA

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