Baculovirus and Insect Cell Expression Protocols

502 Pages·2016·13.73 MB·English

by  David W. Murhammer (eds.)

Methods in
Molecular Biology 1350
David W. Murhammer Editor
Baculovirus
and Insect
Cell Expression
Protocols
Third Edition
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

Baculovirus and Insect Cell
Expression Protocols
Third Edition
Edited by
David W . Murhammer
Department of Chemical & Biochemical Engineering, University of Iowa, Iowa City, IA, USA
Editor
David W. Murhammer
Department of Chemical & Biochemical Engineering
University of Iowa
Iowa City, I A , USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-3042-5 ISBN 978-1-4939-3043-2 (eBook)
DOI 10.1007/978-1-4939-3043-2
Library of Congress Control Number: 2015952020
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Prefa ce
Baculoviruses, which are a group of viruses that infect invertebrates, were fi rst “discovered”
in diseased silk worms in the 1500s, although the viral nature of this disease was not demon-
strated until 1947. Subsequently, hundreds of other baculoviruses have been discovered. For
example, the A utographa californica multiple nucleopolyhedrovirus (AcMNPV) was fi rst
isolated from the alfalfa looper (i.e., A . californica ) insect species. AcMNPV is the most
widely used and best characterized baculovirus and is known to infect many insect species in
addition to A . californica , including S podoptera frugiperda (fall armyworm) and T richoplusia
ni (cabbage looper). Furthermore, AcMNPV is the baculovirus that is usually used to pro-
duce recombinant baculoviruses for subsequent recombinant protein synthesis.
Shangyin Gao and Thomas Grace independently established the fi rst continuous insect
cell lines in the late 1950s and early 1960s. In the 1960s and 1970s insect cell culture was
primarily used as a model to study insect metabolism and for the in vitro synthesis of bacu-
loviruses for potential use in insect control (i.e., as a biopesticide). The widespread use of
insect cell culture, however, did not occur until the baculovirus expression vector system
(BEVS) was independently developed in the Max D. Summers and Lois K. Miller laborato-
ries in the early 1980s. The BEVS takes advantage of the very strong polyhedrin promoter
found in the AcMNPV genome whose natural product (polyhedrin protein) is nonessential
in insect cell culture. Thus, the BEVS involves using the polyhedrin promoter to drive for-
eign protein expression and provides the means to express high levels of recombinant pro-
teins in a relatively short time. Note that there have been many extensions of this basic
principle, many of which are described in this book. In brief, some advantages of the BEVS
are (1) ease of constructing a recombinant baculovirus (compared to isolating stably trans-
formed cells), (2) potentially high expression levels, and (3) the ability of host insect cells
to properly process proteins in a manner similar to mammalian cells. Thus, the BEVS pro-
vides a recombinant protein expression system intermediate between bacteria (e.g., E . coli )
and mammalian cells (e.g., CHO cells) in terms of expression levels, cost, and ability to
perform complex protein modifi cations. The BEVS has become especially popular for
small-scale recombinant protein expression in laboratories throughout the world when bio-
logically active proteins are required for research applications. Furthermore, the BEVS is
becoming a popular choice for producing commercial farm animal and human vaccines. For
example, a subunit/marker vaccine against classical swine fever (“Porcilis Pesti”) produced
by MSD Animal Health was released in 1998 for vaccinating pigs, a virus-like particle vac-
cine against cervical cancer (“Cervarix”) produced by GlaxoSmithKline was released in
2007 for vaccinating human girls, and an annual trivalent fl u vaccine (“Flublok”) produced
by Protein Sciences was released in 2013 for vaccinating humans.
The third edition of B aculovirus and Insect Cell Expression Protocols (the fi rst edition,
edited by Christopher D. Richardson, was published in 1995 and the second edition, edited
by David W. Murhammer, was published in 2007) was written to provide an updated step-
by-s tep guide to biochemists, molecular biologists, biochemical engineers, and others using
the BEVS and/or insect cells for producing recombinant proteins. Furthermore, the third
edition of Baculovirus and Insect Cell Expression Protocols will provide assistance to scientists
v
vi Preface
and engineers interested in developing and producing baculovirus insecticides. In both of
these cases the procedures involved in producing products at laboratory scale and large
scale will be discussed, as well as production in insect larvae.
The third edition of B aculovirus and Insect Cell Expression Protocols is divided into
seven parts. The fi rst part, entitled “Introduction,” contains one chapter that serves as an
overview of the major techniques discussed in detail elsewhere in the book. Furthermore,
this chapter provides step-by-step procedures involved in quantifying cell growth, baculo-
virus infection, and cell metabolism. It is strongly recommended that this chapter be read
prior to reading your specifi c chapter(s) of interest. The second part, entitled “Baculovirus
molecular biology/development of recombinant baculoviruses,” contains three chapters
that give an overview of baculovirus molecular biology and methods involved in construct-
ing and isolating recombinant baculoviruses. Moreover, this part contains another chapter
that discusses using modifi ed baculoviruses to express genes in mammalian cells (“BacMam”).
The third part, entitled “Insect cell culture,” contains four chapters that list currently avail-
able insect cell lines, methods to isolate new cell lines and develop your own serum-free
medium, and routine maintenance and storage of insect cell lines and baculoviruses. The
fourth part, entitled “Protein production with recombinant baculoviruses,” contains four
chapters that discuss small- and large-scale recombinant protein production with the BEVS
in both insect and mammalian cell culture and in insect larvae. The other two chapters in
this part discuss the large-scale production of virus-like particles and an alternative approach
to expressing multicomponent protein complexes. The fi fth part, entitled “Recombinant
protein production with transformed insect cells,” contains a chapter that discusses meth-
ods involved in developing stably transformed insect cells for expressing recombinant pro-
teins directly from the insect cell genome, another chapter about improving the protein
processing capabilities of host insect cells for use with the BEVS, and a chapter about using
Drosophila cell lines, which provide an alternative to the lepidopteran insect cell lines used
with the BEVS. The sixth part, entitled “Baculovirus development and production for use
as insecticides,” contains three chapters about the use, production, and characterization of
baculoviruses (both wild type and recombinant) for use as biopesticides. The seventh part,
entitled “Miscellaneous techniques and applications of the baculovirus/insect cell system,”
contains three chapters that discuss the use of green fl uorescent protein, tubular reactors,
and RNAi for research applications. The other two chapters discuss the application of the
baculovirus/insect cell system to study apoptosis and generating envelop-modifi ed baculo-
virus for gene delivery into mammalian cells.
The third edition of B aculovirus and Insect Cell Expression Protocols provides the detailed
steps required to perform the techniques involved with the use of baculoviruses and insect
cell culture and discusses problems that may be encountered. It is hoped that this book will
not only aid the user in successfully completing the tasks described herein but also stimulate
the development of improved techniques and new applications of baculoviruses and insect
cell culture.
The editor would like to thank the 39 contributors for their excellent submissions and
the Series Editor, John Walker, for his guidance throughout the process of producing this
book. Furthermore, a special thanks to Katie Schnedler for her assistance in converting
fi gures into the proper format.
Iowa City, IA, USA D avid W. Murhammer
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i
PART I INTRODUCTION
1 Useful Tips, Widely Used Techniques, and Quantifying
Cell Metabolic Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
David W . M urhammer
PART II B ACULOVIRUS MOLECULAR BIOLOGY/DEVELOPMENT
OF RECOMBINANT BACULOVIRUSES
2 Introduction to Baculovirus Molecular Biology. . . . . . . . . . . . . . . . . . . . . . . . 2 5
Barbara J. K elly , Linda A . K ing , and Robert D. P ossee
3 B aculovirus Transfer Vectors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Robert D . P ossee and Linda A . K ing
4 Recombinant Baculovirus Isolation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Linda A . K ing , Richard H itchman , and Robert D. Possee
5 Gene Expression in Mammalian Cells Using BacMam,
a Modified Baculovirus System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 5
James A. F ornwald , Quinn L u , Frederick M. Boyce , and Robert S. A mes
PART III INSECT CELL CULTURE
6 Available Lepidopteran Insect Cell Lines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Dwight E. Lynn and R obert L . H arrison
7 L epidopteran Insect Cell Line Isolation from Insect Tissue . . . . . . . . . . . . . . . 1 43
Dwight E . L ynn
8 D evelopment of Serum-Free Media for Lepidopteran Insect Cell Lines. . . . . . 1 61
Leslie C. L. Chan and Steven R eid
9 R outine Maintenance and Storage of Lepidopteran Insect Cell Lines
and Baculoviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 97
Dwight E . L ynn and Robert L . H arrison
PART IV PROTEIN PRODUCTION WITH RECOMBINANT BACULOVIRUSES
10 Small-Scale Production of Recombinant Proteins Using the Baculovirus
Expression Vector System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 25
Jian-Ping Yang
11 Recombinant Protein Production in Large-Scale Agitated Bioreactors
Using the Baculovirus Expression Vector System. . . . . . . . . . . . . . . . . . . . . . . 241
Christine M . T hompson , Johnny M ontes , Marc G. A ucoin ,
and Amine A. Kamen
vii
viii Contents
12 Protein Expression in Insect and Mammalian Cells Using Baculoviruses
in Wave Bioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Sue H . Kadwell and Laurie K . Overton
13 P rotein Production with Recombinant Baculoviruses
in Lepidopteran Larvae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Elena Kovaleva and David C. D avis
14 P roduction of Virus-Like Particles for Vaccination. . . . . . . . . . . . . . . . . . . . . . 2 99
Christine M. Thompson , M arc G. A ucoin , and Amine A. Kamen
15 A lternative Strategies for Expressing Multicomponent Protein
Complexes in Insect Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Stephanie C hen
PART V RECOMBINANT PROTEIN PRODUCTION WITH TRANSFORMED
INSECT CELLS
16 Transforming Lepidopteran Insect Cells for Continuous Recombinant
Protein Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 29
Robert L. Harrison and Donald L . J arvis
17 Stable Drosophila Cell Lines: An Alternative Approach
to Exogenous Protein Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 49
Marija B ackovic and T homas K rey
18 T ransforming Lepidopteran Insect Cells for Improved Protein
Processing and Expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
Robert L . H arrison and D onald L . J arvis
PART VI BACULOVIRUS DEVELOPMENT AND PRODUCTION
FOR USE AS INSECTICIDES
19 Introduction to the Use of Baculoviruses as Biological Insecticides . . . . . . . . . 3 83
Holly J. R . Popham , Tyasning N usawardani , and Bryony C. Bonning
20 Baculovirus Insecticide Production in Insect Larvae . . . . . . . . . . . . . . . . . . . . 3 93
Nikolai v an Beek and David C . Davis
21 E valuation of the Insecticidal Efficacy of Wild Type
and Recombinant Baculoviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
Holly J. R . P opham , Mark R. Ellersieck , Huarong L i ,
and Bryony C. B onning
PART VII MISCELLANEOUS TECHNIQUES AND APPLICATIONS
OF THE BACULOVIRUS/INSECT CELL SYSTEM
22 Evaluating Baculovirus Infection Using Green Fluorescent
Protein and Variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 47
Hsuan-Chen Wu , H yung J oon C ha , and W illiam E. Bentley
23 T ubular Bioreactor for Probing Baculovirus Infection
and Protein Production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461
Hsuan-Chen W u , Yu-Chen Hu , and William E. Bentley
Contents ix
24 Gene Silencing in Insect Cells Using RNAi. . . . . . . . . . . . . . . . . . . . . . . . . . . 4 69
Hsuan-Chen Wu , J ohn C. M arch , and William E. B entley
25 U sing the Baculovirus/Insect Cell System to Study Apoptosis. . . . . . . . . . . . . 4 77
Nor Chejanovsky
26 G eneration of Envelope-Modified Baculoviruses for Gene
Delivery into Mammalian Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
Christian Hofmann
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 05