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The Baculovirus Expression System
The Baculovirus Expression
System
The Baculovirus Expression
System A laboratory guide
L A. King School of Biological and Molecular Sciences,
Oxford Polytechnic, Oxford, UK
R. D. Possee Natural Environment Research Council,
Institute of Virology and Environmental Microbiology, Oxford, UK
m SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.
First edi t ion 1992
© 1992 L . A . K i n g and R. D . Possee Originally published by Chapman & H a l l in 1992
Typeset i n IO/IIV2 Palatino by Falcon Typographic A r t L t d , E d i n b u r g h
ISBN 978-94-010-5047-0 I S B N 978-94-011-2374-7 (eBook) D O I 10.1007/978-94-011-2374-7
Apar t from any fair dealing for the purposes of research or private study, or cr i t ic ism or review, as permitted under the U K Copyr ight Designs and Patents A c t , 1988, this publ icat ion may not be reproduced, stored, or transmitted, i n any form or by any means, without the pr ior permiss ion i n w r i t i n g of the publishers , or i n the case of reprographic reproduction only i n accordance w i t h the terms of the licences by the Copyr ight L icens ing Agency i n the U K , or i n accordance w i t h the terms of licences issued by the appropriate Reproduct ion Rights Organizat ion outside the U K . Enquir ies concerning reproduction outside the terms stated here should be sent to the publishers at the L o n d o n address pr inted on this page.
The publ isher makes no representation, express or i m p l i e d , w i t h regard to the accuracy of the information contained i n this book and cannot accept any legal responsibi l i ty or l iab i l i ty for any errors or omissions that may be made.
A catalogue record for this book is available from the Bri t ish Library
L ibrary of Congress Cataloging- in-Publ icat ion data available
For Simon David
Contents
1 The baculoviruses 1 1.1 Introduction 1 1.2 Isolation and host range 1 1.3 Structure and classification 2 1.4 Baculovirus replication in vivo 4 1.5 Baculovirus replication in vitro 7
1.5.1 Baculovirus gene expression and replication 8 1.5.2 Baculovirus gene promoters 12
1.6 Genetic engineering of baculovirus insecticides 15
2 The development of baculovirus expression vectors 16 2.1 Introduction and historical perspective 16 2.2 The merits of the baculovirus expression system 17
2.2.1 Advantages 17 2.2.2 Disadvantages 18
2.3 General principles for inserting foreign genes into the baculovirus genome 18
2.4 Baculovirus transfer vectors 19 2.4.1 Polyhedrin promoter-based expression vectors 21 2.4.2 p10 promoter-based transfer vectors 25 2.4.3 Multiple expression vectors 27 2.4.4 Transfer vectors utilizing other baculovirus
gene promoters 29 2.5 Selection of recombinant viruses 30
2.5.1 Selection of a polyhedrin-negative phenotype 31 2.5.2 Selection of f3-galactosidase-negative viruses 33
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2.5.3 Recombinant virus selection using dot-blot hybridization 33
2.5.4 Screening for a positive phenotype 34 2.5.5 Enhancing the numbers of recombinant viruses 34
3 Processing of foreign proteins synthesized using baculovirus vectors in insect cells 37 3.1 Introduction 37 3.2 Glycosylation 39 3.3 Phosphorylation, acylation and amidation 42 3.4 Proteolytic processing 44 3.5 Cellular targeting and secretion 45 3.6 Tertiary and quaternary structure formation 47 3.7 Expression of viral genes 48 3.8 Expression of bacterial and fungal genes 48 3.9 Post-transcriptional processing 50
4 Construction of transfer vectors containing the foreign gene 51 4.1 Introduction 51 4.2 Isolation of foreign gene coding sequences 51
4.2.1 Some general guidelines 51 4.2.2 Isolation of DNA fragments from agarose gels 53
4.3 Modifying the ends of DNA molecules 55 4.3.1 Mung bean nuclease 55 4.3.2 Klenow fill-in 55
4.4 Preparation of the transfer vector 4.5 DNA ligations 4.6 Transformation of bacteria 4.7 Screening for recombinant baculovirus transfer
vectors 4.7.1 Colony hybridization 4.7.2 Rapid isolation of bacterial plasmid
DNA (mini-preps) 4.8 Analysis of recombinant transfer vectors 4.9 Isolation of highly purified plasmid DNA
(maxi-preps)
56 57 58
60 60
62 63
72
5 Insect cell culture media and maintenance of insect cell lines 75 5.1 Introduction 75 5.2 Cell lines 75 5.3 Culture media 76 5.4 Preparation of culture media 79
5.4.1 Preparation of TC100lFCS growth medium 79 5.4.2 Preparation of Grace's (TNM-FH) growth medium 80 5.4.3 Preparation of 4.5 1 TC100 medium from
powdered formula 81
Contents / ix
5.4.4. Preparation of TC100 medium from individual ingredients 83
5.4.5 Specialized TC100 media 87 5.4.6 Alternative insect cell culture media 87
5.5 Glassware and disposable plasticware 90 5.5.1 Suggested cleaning regime for tissue culture
glassware 90 5.6 Insect cell culture 91
5.6.1 Routine sub-culturing of Sf cell lines (monolayer cultures) 93
5.6.2 Routine sub-culturing of Sf cells maintained in spinner cultures 98
5.7 A guide to Sf cell seeding densities for experimental work 102
5.8 Freezing, storage and recovery of insect cells in liquid nitrogen 102 5.8.1 Freezing and storage of cells in liquid nitrogen 102 5.8.2 Recovery of cells from liquid nitrogen 104
5.9 A guide to adapting cells to serum-free media 104
6 Propagation, titration and purification of AcMNPV in cell culture 106 6.1 Introduction 106
6.1.1 Safety considerations: general rules for working with baculoviruses 106
6.2 Infection of cells with virus for experimental work 108 6.2.1 Infection of Sf cells in monolayer culture 108 6.2.2 Infection of Sf cells in suspension culture 110
6.3 Titration of virus by plaque-assay 111 6.3.1 Standard plaque-assay 111 6.3.2 Plaque-assay of lacZ-positive viruses 114
6.4 Plaque-picking and plaque-purification 115 6.5 Amplification of virus stocks 116
6.5.1 To prepare a seed stock of virus from a plaque-pick 116
6.5.2 Preparation of an intermediate stock of virus 117 6.5.3 Preparation of a high-titre working stock of virus 118
6.6 Large-scale production of virus for the purification of virus particles 119
6.7 Purification of infectious virus DNA 121 6.8 Titration of virus by TCID50 124
7 Production and selection of recombinant virus 127 7.1 Introduction 127
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7.2 Preparation of linear AcMNPY.ZacZ (or AcMNPY.SC) DNA 129
7.3 Co-transfection of insect cells 132 7.3.1 Co-transfection by lipofection 132 7.3.2 Co-transfection by calcium phosphate
co-precipitation 133 7.4 Separation of parental and recombinant viruses by
plaque-assay 135 7.5 Plaque-purification and amplification of recombinant
virus stocks 138 7.6 Amplification and detection of recombinant viruses
by limiting dilution and dot-blot hybridization 138
8 Characterization of recombinant viruses 141 8.1 Introduction 141 8.2 Analysis of recombinant virus genomes 142
8.2.1 Extraction of DNA from virus-infected cells 143 8.2.2 Analysis of virus DNA with restriction
endonucleases 144 8.2.3 Southern hybridization analysis of virus DNA 146
8.3 Analysis of foreign gene expression by polyacrylamide gel electophoresis, using unlabelled or radiolabelled cell proteins 148 8.3.1 Radiolabelling proteins in virus-infected
insect cells 149 8.3.2 Polyacrylamide gel electrophoresis of infected
cell extracts 152 8.4 Analysis of recombinant protein synthesis in
insect cells using immunological techniques 154 8.4.1 Immunofluorescence 154 8.4.2 Western blot analysis of virus-infected
cell proteins 156 8.4.3 Immunoprecipitation of virus-infected cell
proteins 158 8.5 Analysis of post-translational processing events
in insect cells 161 8.5.1 Glycosylation 162 8.5.2 Phosphorylation 163 8.5.3 Palmitylation and myristylation 163
8.6 Analysis of transcription in recombinant virus-infected cells 164 8.6.1 Extraction of RNA from insect cells 165 8.6.2 Analysis of RNA using Northern blot
hybridization 167
Contents / xi
9 Scaling up the production of recombinant protein in insect cells; laboratory bench level 171 9.1 Introduction 171 9.2 Large-scale culture of insect cells 173
9.2.1 Large-scale culture of insect cells in monolayer cultures 173
9.2.2 Large-scale culture of insect cells in suspension cultures 174
9.3 The importance of highly infectious virus stocks 176 9.4 Multiplicity of infection 177 9.5 The optimum time to harvest virus-infected cells 177 9.6 Purification of recombinant protein from infected
rellrultures 1~
10 Propagation of baculoviruses in insect larvae 180 10.1 Introduction 180 10.2 Rearing insects in the laboratory 181 10.3 Infection of insect larvae with polyhedra from cell
rulture 182 10.3.1 Preparation of virus polyhedra from infected
cells in rulture 182 10.3.2 Propagating the virus in insect larvae 183
10.4 Purification of polyhedra from infected larvae 184 10.5 Bioassays of polyhedra 187
10.5.1 LDso assays 187 10.5.2 LTso assays 188
10.6 Purification of virus particles and DNA from polyhedra 188
10.7 Isolation of virus particles from infected larvae to establish infections in cell culture 189 10.7.1 Purification of virus particles from polyhedra for
the infection of cells in culture 191 10.7.2 Purification of virus particles from haemolymph
for the infection of cells in culture 191 10.8 Preparation of semi-synthetic insect diet 192
11 Trouble-shooting guide 195 11.1 Introduction 195 11.2 Insertion of foreign gene coding sequences into
transfer vectors 195 11.2.1 The transfer vector 195 11.2.2 DNA sequences for insertion into transfer
vectors 196 11.2.3 Ligations 197
11.3 Cell culture 198
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11.3.1 Cells fail to thrive and attach to glass/plastic surfaces 198
11.3.2 Cells are contaminated with virus 199 11.3.3 Cells are contaminated with yeast, fungi
or bacteria 199 11.3.4 Crystals of precipitate in the medium 200
11.4 Preparation of virus stocks and infectious DNA 200 11.4.1 Virus stocks 200 11.4.2 Infectious virus DNA purification 200
11.5 Co-transfections 201 11.6 Baculovirus plaque-assays 201
11.6.1 Condition of the cells 201 11.6.2 Plaque-assay manipulations 202 11.6.3 General problems 202
11.7 Screening for recombinant viruses 203 11.8 Instability of recombinant viruses 204 11.9 Poor yields of recombinant protein 204
Appendix A list of selected suppliers 206
References 210
Index 222
Preface
The decision to write a book about the practical aspects of the baculovirus expression system stems from the numerous phone calls for help we have had, and from the many visitors to our laboratories requiring assistance to find the elusive polyhedrin-negative virus containing their favourite gene. We have also organized two expression system workshops and from the manuals we wrote for these, it seemed a logical progression to extend them into book form. We appreciate that those who are 'old-hands' at the baculovirus expression system may have differing views on some of our procedures, but the methods in this book are presented in the light of our own experiences in the laboratory and from our practical workshops, and we hope that the book will be especially useful to those new to the system.
The first three chapters give the background information to the baculovirus expression system, and includes advice on how to choose the right transfer vector and discusses the various methods that are available to select recombinant viruses. The practical chapters concentrate on those aspects which are novel to the baculovirus system (insect cell culture, virus amplification and titration, etc.) and, in general, leave the standard molecular biological techniques to the other excellent laboratory manuals that are available. However, for completeness sake and to avoid constant reference to other manuals, we have included brief details of some standard techniques where they are integral to the success of the baculovirus protocols.
Many people have wittingly, or unwittingly, contributed to the preparation of this book. We cannot thank them all by name but wish them to know that we are grateful for the help received. In particular we would like to thank the past and present members of our research groups; at the Polytechnic, Susan Mann, Alison Lawrie, Dr Susan Marlow, Kate Trapnell, Dr Matthew Weitzman, Dr Allan Atkinson, Louis Obosi, David Hughes, Chris Palmer, Kirsti Joyce,
xiv / Preface
Davin Miller and Dr Jason Pickering; and at IVEM, Dr Ulrike Weyer, Dr Paul Kitts, Dr Alison Clarke (nee Merryweather), Dr Lorna Stewart, Dr Katy Gearing, Dr Stuart Knight, Steve Howard, Martin Ayres, Jan Pullen, Rachael Hawtin and Dr Miguel Lopez-Ferber. Many of the techniques described in this book have been 'tried and tested' by the above. We would also like to thank Karin Chaloner at IVEM for passing on her expertise in preparing insect cell culture media. We would particularly like to acknowledge the inspiration of Paul Kitts and Martin Ayres, who provided the insight for the linear DNA technique described in Chapter 4. This methid, more than any other, has revolutionized the production of recombinant baculoviruses.
We would also like to thank Derek Whitely of Oxford Polytechnic for his excellent line drawings (Figures 1.1, 1.2, 5.1 and 5.3) and for preparing the drawing on the front cover, and Barbara Southall, Susan Marlow and Chris Hatton for help with some of the photography.
We are grateful to Dr Susan Hemmings and Philippa MacBain, and others in the sub-editorial and production departments at Chapman & Hall for their encouragement and guidance, and for keeping us just about on time.
Finally, we are grateful to our son, Simon, for being a well-behaved baby and allowing us to continue writing and editing!
Linda A. King Robert D. Possee
