Abstract
With an increasing need for functional analysis of proteins, there is a growing demand for fast and cost-effective production of biologically active eukaryotic proteins. The baculovirus expression vector system (BEVS) is widely used, and in the vast majority of cases cultured insect cells have been the host of choice. A low cost alternative to bioreactor-based protein production exists in the use of live insect larvae as “mini bioreactors.” In this chapter we focus on Trichoplusia ni as the host insect for recombinant protein production, and explore three different methods of virus administration to the larvae. The first method is labor-intensive, as extracellular virus is injected into each larva, whereas the second lends itself to infection of large numbers of larvae via oral inoculation. While these first two methods require cultured insect cells for the generation of recombinant virus, the third relies on transfection of larvae with recombinant viral DNA and does not require cultured insect cells as an intermediate stage. We suggest that small- to mid-scale recombinant protein production (mg-g level) can be achieved in T. ni larvae with relative ease.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Maeda S, Kawai T, Obinata M et al (1985) Production of human α-interferon in silkworm using a baculovirus vector. Nature 315:592–594
Miyajima A, Schreurs J, Otsu K et al (1987) Use of the silkworm, Bombyx mori, and an insect baculovirus vector for high-level expression and secretion of biologically active mouse interleukin-3. Gene 58:273–281
Zhu J, Ze Y, Zhang C et al (2006) High-level expression of recombinant human paraoxonase 1 Q in silkworm larvae (Bombyx mori). Appl Microbiol Biotechnol 72:103–108
Du D, Kato T, Suzuki F et al (2009) Expression of protein complex comprising the human prorenin and (pro)renin receptor in silkworm larvae using bombyx mori nucleopolyhedrovirus (BmNPV) bacmids for improving biological function. Mol Biotechnol 43:154–161
Usami A, Ishiyama S, Enomoto C et al (2011) Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf-9) and silkworm. J Biochem 149:219–227
Gretch D, Sturley S, Friesen P et al (1991) Baculovirus-mediated expression of human apolipoprotein E in Manduca sexta larvae generates particles that bind to the low density lipoprotein receptor. Proc Natl Acad Sci U S A 88:8530–8533
Hellers M, Steiner H (1992) Diapausing pupae of Hyalophora cecropia: an alternative host for baculovirus mediated expression. Insect Biochem Mol Biol 22:35–39
Ahmad S, Bassiri M, Banerjee A et al (1993) Immunological characterization of the VSV nucleocapsid (N) protein expressed by recombinant baculovirus in Spodoptera exigua larvae: use in differential diagnosis between vaccinated and infected animals. Virology 192:207–216
Kuroda K, Groener A, Frese K et al (1989) Synthesis of biologically active influenza virus hemagglutinin in insect larvae. J Virol 63:1677–1685
Richardson C, Banville M, Lalumiere M et al (1992) Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae. Intervirology 34:213–227
Argaud O, Croizier L, Lopez-Ferber M et al (1998) Two key mutations in the host-range specificity domain of the p143 gene of Autographa californica nucleopolyhedrovirus are required to kill Bombyx mori larvae. J Gen Virol 79:931–935
Groener A (1989) Host range of AcNPV. In: Granados R, Federici B (eds) The biology of baculoviruses, vol 1. CRC, Boca Raton, FL, pp 177–188
Kovaleva E, O’Connell K, Buckley P et al (2009) Recombinant protein production in insect larvae: host choice, tissue distribution and heterologous gene instability. Biotechnol Lett 31:381–386
Villalba M, Wente S, Russell D et al (1989) Another version of the human insulin receptor kinase domain: expression, purification, and characterization. Proc Natl Acad Sci U S A 86:7848–7852
Medin J, Hunt L, Gathy K et al (1990) Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae. Proc Natl Acad Sci U S A 87:2760–2764
Tremblay N, Kennedy B, Street I et al (1993) Human group II phospholipase A2 expressed in Trichoplusia ni larvae—isolation and kinetic properties of the enzyme. Protein Expr Purif 4:490–498
Pham M-Q, Naggie S, Wier M et al (1999) Human interleukin-2 production in insect (Trichoplusia ni) larvae: effects and partial control of proteolosis. Biotechnol Bioeng 62:175–182
Hale C, Zimmerschied J, Bliler S et al (1999) Large-scale expression of recombinant cardiac sodium-calcium exchange in insect larvae. Protein Expr Purif 15:121–126
O’Connell K, Kovaleva E, Campbell J et al (2007) Production of a recombinant antibody fragment in whole insect larvae. Mol Biotechnol 36:44–51
Rubiolo J, López-Alonso H, Alfonso A et al (2012) Characterization and activity determination of the human protein phosphatase 2A catalytic subunit α expressed in insect larvae. Appl Biochem Biotechnol 167:918–928
Gómez-Casado E, Gómez-Sebastian S, Núñez M et al (2011) Insect larvae biofactories as a platform for influenza vaccine production. Protein Expr Purif 79:35–43
Perez-Filgueira M, Resino-Talaván P, Cubillos C et al (2007) Development of a low-cost, insect larvae-derived recombinant subunit vaccine against RHDV. Virology 364:422–430
Millán A, Gómez-Sebastián S, Nuñez M et al (2010) Human papillomavirus-like particles vaccine efficiently produced in a non-fermentative system based on insect larva. Protein Expr Purif 74:1–8
Pérez-Marín E, Gómez-Sebastián S, Argilaguet J et al (2010) Immunity conferred by an experimental vaccine based on the recombinant PCV2 Cap protein expressed in Trichoplusia ni-larvae. Vaccine 28:2340–2349
Greenblatt H, Otto T, Kirkpatrick M et al (2012) Structure of recombinant human carboxylesterase 1 isolated from whole cabbage looper larvae. Acta Crystallogr Sect F Struct Biol Cryst Commun 68:269–272
Lihoradova O, Ogay I, Abdukarimov A et al (2007) The Homingbac baculovirus cloning system: an alternative way to introduce foreign DNA into baculovirus genomes. J Virol Methods 140:59–65
Wood H, Trotter K, Davis T et al (1993) Per os infectivity of preoccluded virions from polyhedrin-minus recombinant baculoviruses. J Invertebr Pathol 62:64–67
Wood HA (1997) Stable pre-occluded virus particle. US Patent 5,593,669
Wood HA (2000) Stable pre-occluded virus particle for use in recombinant protein production and pesticides. US Patent 6,090,379
O’Reilly D, Brown M, Miller L (1992) Alteration of ecdysteroid metabolism due to baculovirus infection of the fall armyworm, Spodoptera frugiperda: host ecdysteroids are conjugated with galactose. Insect Biochem Mol Biol 22:313–320
Baird G, Zacharias D, Tsien RY (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A 97:11984–11989
Wu X, Cao C, Kumar V et al (2004) An innovative technique for inoculating recombinant baculovirus into the silkworm Bombyx mori using lipofectin. Res Microbiol 155:462–466
Acknowledgement
We thank Ian Smith (Nara Institute of Science and Technology, Japan) for reviewing the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Kovaleva, E., Davis, D.C. (2016). Protein Production with Recombinant Baculoviruses in Lepidopteran Larvae. 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_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3043-2_13
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3042-5
Online ISBN: 978-1-4939-3043-2
eBook Packages: Springer Protocols