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Transforming Lepidopteran Insect Cells for Continuous Recombinant Protein Expression

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Baculovirus and Insect Cell Expression Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1350))

Abstract

The baculovirus expression vector system (BEVS) is widely used to produce large quantities of recombinant proteins. However, the yields of extracellular and membrane-bound proteins obtained with this system are often very low, possibly due to the adverse effects of baculovirus infection on the host insect cell secretory pathway. An alternative approach to producing poorly expressed proteins is to transform lepidopteran insect cells with the gene of interest under the control of promoters that are constitutively active in uninfected cells, thereby making cell lines that continuously express recombinant protein. This chapter provides an overview of the methods and considerations for making stably transformed lepidopteran insect cells. Techniques for the insertion of genes into continuous expression vectors, transfection of cells, and the selection and isolation of stably transformed Sf-9 clones by either colony formation or end-point dilution are described in detail.

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Acknowledgments

D.L.J. gratefully acknowledges the NIH (GM49734), the NSF (BES-9814157 and BES-9818001), and the USDA-NRI (89-37266-4935 and 95-37302-1921/2658) for supporting work in his lab. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

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Authors and Affiliations

  1. Invasive Insect Biocontrol & Behavior Laboratory, USDA, ARS, BARC, Building 007, Room 301, BARC-W, 10300 Baltimore Avenue, Beltsville, MD, 20705, USA

    Robert L. Harrison

  2. Department of Molecular Biology, University of Wyoming, Laramie, WY, USA

    Donald L. Jarvis

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  1. Robert L. Harrison
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  2. Donald L. Jarvis
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Correspondence to Robert L. Harrison .

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Editors and Affiliations

  1. Engineering, University of Iowa Dept. Chemical & Biochemical, Iowa City, Iowa, USA

    David W. Murhammer

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Harrison, R.L., Jarvis, D.L. (2016). Transforming Lepidopteran Insect Cells for Continuous Recombinant Protein Expression. In: Murhammer, D. (eds) Baculovirus and Insect Cell Expression Protocols. Methods in Molecular Biology, vol 1350. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3043-2_16

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  • DOI: https://doi.org/10.1007/978-1-4939-3043-2_16

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3042-5

  • Online ISBN: 978-1-4939-3043-2

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