Cell Engineering: Transient Expression

229 Pages·2000·16.97 MB·English

by  T. A. Kost

Cell Engineering
Cell Engineering
Volume 2
Editor-in-Chief
Dr Mohamed Al-Rubeai
School of Chemical Engineering,
The University of Birmingham, Edgbaston, Birmingham, U.K.
Editorial Board
Dr Hansjorg Hauser
GBF,
Braunschweig, Germany
Professor Michael Betenbaugh
Johns Hopkins University,
Baltimore, U.S.A.
Dr Nigel Jenkins
Bioprocess Research & Development,
Lilly Research Laboratories, Indianapolis, U.S.A.
Professor Caroline MacDonald
University of Paisley,
Paisley, U.K.
Dr Otto-Wilhelm Merten
A.EM.-Genethon 11,
Gene Therapy Program,
Evry, France
The titles published in this series are listed at the end of this volume
CELL ENGINEERING
Vol. 2: Transient Expression
Edited by
Mohamed Al-Rubeai
School of Chemical Engineering,
The University of Birmingham,
Edgbaston, Birmingham, U.K.
SPRTNGER-SCIENCE+BUSINESS MEDIA, B.V.
Library of Congress Cataloging-in-Publication Data
ISBN 978-94-010-5866-7 ISBN 978-94-011-4315-8 (eBook)
DOI 10.1007/978-94-011-4315-8
Printed on acid-free paper
All Rights Reserved
© 2000 Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 2000
Softcover reprint of the hardcover 1st edition 2000
No part of the material protected by this copyright notice may be reproduced or
utilized in any form or by any means, electronic or mechanical,
including photocopying, recording or by any information storage and
retrieval system, without written permission from the copyright owner.
Transient Expression
Cell Engineering
Volume 2
With contributions by
E. Ailor
A. R. Bernard
M. J. Betenbaugh
1. P. Condreay
A. M. Douar
R. F. Jannan
M. Jordan
1. L. Klein
T. A. Kost
P. Liljestrom
C. Mannix
B. Massie
W. Oualikene
C. Smerdou
Contents
APPLICATION OF RECOMBINANT BACULOVIRUSES IN
BIOPHARMACEUTICAL RESEARCH
T.A.Kost, lL. Klein and J.P.Condreay
ENGINEERING POST-TRANSLATIONAL PROCESSING OF 29
RECOMBINANT PROTEINS PRODUCED IN INSECT CELL CULTURE
E. Ailor and M.l Betenbaugh
A GUIDE TO SUCCESSFUL SCALE-UP OF THE BACULOVIRUS 43
EXPRESSION SYSTEM
C. Mannix and R.F. Jannan
TRANSIENT GENE EXPRESSION IN MAMMALIAN CELLS BASED ON 56
THE CALCIUM PHOSPHATE TRANSFECTION METHOD
M. Jordan
ADENOVIRUS VECTORS IN FUNCTIONAL GENOMICS 80
W. Oualikene and B. Massie
ADENO-ASSOCIATED VIRUS: A PROMISING TOOL FOR GENE 155
DELIVERY
A.M. Douar
ALPHA VIRUS VECTORS: HIGHLY EFFICIENT SYSTEMS FOR 182
TRANSIENT GENE EXPRESSION
C. Smerdou and P. Liljestrom
TRANSIENT EXPRESSION IN MAMMALIAN CELLS: APPLICATIONS 211
AND PERSPECTIVES
A.R. Bernard
Subject Index 219
APPLICATION OF RECOMBINANT BACULOVIRUSES IN
BIOPHARMACEUTICAL RESEARCH
T. A. KOST*, J. L. KLEIN AND J. P. CONDREAY
Protein Sciences and BioMetabolism Departments, Glaxo Wellcome
Research and Development, 5 Moore Drive, Research Triangle Park,
North Carolina 27709 US.A.
*Corresponding author, fax [phone]
e-mail [email protected]
1. Introduction
The first papers describing the use of insect cell baculoviruses as expression vectors
were published in the early 1980's (Smith et al., 1983; Pennock et al., 1984).
Interestingly, as measured by the number of published papers identified using the terms
"recombinant and baculovir*" to search the PubMed database, the system had only
limited use through 1990 (Fig. 1). One reason for the lack of utilization over this period
may have been due to the tedious procedures initially required to identify and isolate
recombinant baculoviruses (rBV). The recombination frequency of the original
baculovirus expression vector system (BEVS) was very low, less than 1% . This low
recombination frequency made it difficult for those unskilled in the art to identify and
isolate purified recombinant viruses. In the 1990' s the development of a variety of new
transfer plasmids (Jarvis, 1997), linearized, gapped viral DNA's (Kitts et al., 1990; Kitts
and Possee, 1993) and a novel system based on site-specific transposition in E. coli
(Luckow et al., 1993) greatly simplified and rapidly expanded the use of the system. In
addition, the reagents required for generating recombinant viruses became more widely
available through commercial sources.
A number of attributes, highlighted in Table 1, make the BEVS system particularly
attractive for the small and large scale production of recombinant proteins in insect cells
(Miller, 1997; Vlak et al., 1996). In order to compensate for the potential disadvantages
of the system, a variety of approaches are being investigated to overcome the
glycosylation deficiencies of insect cells as compared to mammalian cell lines (Jarvis et
al., 1998; Altmann et aI., 1999). Non lytic insect cell based systems have also been used
successfully for the stable production of recombinant proteins (McCarroll and King,
1997; Pfeifer, 1998); however, these approaches will not be discussed in this chapter.
In addition to their application for the production of recombinant proteins in infected
insect cell and larvae hosts, (rBV) have been modified to direct transient and stable
expression of recombinant proteins in mammalian cells, as reviewed by Kost and
Condreay (1999). rBV containing mammalian viral and cellular promoter elements have
been shown to efficiently transduce a wide variety of mammalian cell types without
M. Al-Rubeai (ed.), Cell Engineering, 1-28.
© 2000 Kluwer Academic Publishers.
2
discernable cytopathology. This provides a unique system, whereby one can easily
generate and produce the desired recombinant viruses in the natural insect cell host and
then use them to transduce mammalian cells in the absence of viral replication.
j
700
i~
'0
~ 400 o
~
3X)
200
100
y .....
Figure 1. Publications utilizing rBY.
Another emerging use of rBV is that of virus display technology, first reported by
Boublik et al. in 1995. A protein as large and complex as human immunodeficiency
virus gp120 has been successfully displayed on the surface ofbaculovirus particles.
TABLE I. Advantages and disadvantages of the BEYS for recombinant protein production in insect cells.
Advantages Disadvantages
• Ease of use • Lytic system
• Wide variety of transfer vectors • Protein glycosylation patterns
• High expression level potential
• Functional protein production
• Production of multi-subunit complexes
• Proven biosafety profile
• Anchorage dependent or suspension culture cell growth
• Cell growth in serum free medium
• Ease of scale-up
The sequence of the human genome is nearly complete. The human genome is
predicted to encode upwards of 100,000 gene products. Undoubtedly rBV, as well as
other viral vector systems, will serve as useful tools for the production and study of
many of these proteins. The intent of this chapter is to provide a brief overview of
baculovirus biology, vector technology and its application for recombinant protein
production in insect and mammalian cells. Insect cell applications will focus on the
3
production of multi-subunit protein complexes, G-protein coupled receptors, ion
channels and cytochrome P450s. The more recent application of modified recombinant
baculoviruses for transient and stable protein production in mammalian cells and
developments in the application ofbaculovirus display will also be described.
2. Baculovirus Biology
Baculoviridae comprise a family of lytic viruses that are pathogens of a wide variety of
arthropods. Study of the granulovirus genus of theses viruses is hampered by a lack of
cell culture systems to propagate the virus (Kelly, 1982). In contrast, much is known
about the biology of the nucleopolyhedrosisviruses, including the prototype of this
genus, Autographa califa rnica nuclear polyhedrosis virus (AcMNPV). AcMNPV can be
efficiently propagated in vivo and in vitro in cultured cell lines derived from insect
hosts. A collection of recent reviews of baculoviruses can be found in Miller (1997),
however some relevant aspects of AcMNPV biology will be discussed as they relate to
the development of this virus as an expression vector.
The genome of these viruses is a double-stranded circular DNA ranging in size from
90 to 160 kb. Currently, in addition to AcMNPV (Accession number L22858; Ayers et
al., 1994), the complete nucleotide sequences of four other baculoviruses (Accession
numbers AF169823, L33180, AF08181O, and U75930) have been determined. The
AcMNPV genome is 133,894 bp and is predicted to contain approximately 150 open
reading frames dispersed througout the genome on both strands. The complete pattern
of all expressed genes and their functions remain to be fully elucidated.
Baculovirus genes are grouped into classes depending upon their time of expresssion
after infection (Kool and Vlak, 1993). The immediate early genes do not require viral
gene expression to be transcribed, and thus, transcriptional regulatory regions from
these genes have found utility as promoters for the engineering of stably transfected
insect cell lines (McCarroll and King, 1997; Pfeifer, 1998). However, transcription of
successive groups of genes require products from the previous group for their
expression (Miller, 1996). Late and very late genes contain a consensus transcriptional
start site, the TAAG motif (Blissard and Rohrmann, 1990). Promoters from these highly
regulated genes, that are used to direct expression of foreign genes in rBV, are not
expressed in uninfected insect cells (Miller, 1996), or in other cell types in which the
virus does not replicate (Carbonell et al., 1985; unpublished data from our laboratory).
Baculoviruses take on one of two morphological forms that fulfill different roles in
the dual life cycle of the virus (Miller, 1996). Both forms of the virus contain the same
nucleocapsid structure and genetic complement. Occlusion-derived virus (ODV)
consists of one or more membrane-enclosed nucleocapsids which are embedded in a
protein matrix forming a polyhedron. This form of the virus is responsible for infection
of the insect host in the wild. The protein matrix of the polyhedron is comprised largely
of the product of the viral polyhedrin protein gene (Rohrmann, 1986), the single most
highly expressed viral gene product (Jarvis, 1997). The production of polyhedra or
occluded virus occurs late in infection.
Early in infection, nucleocapsids migrate out of the nucleus where the virus
replicates and bud through the cytoplasmic membrane of the cell (Fraser, 1986). As it
4
exits the cell the budded form of the virus picks up an envelope with spike structures at
one end (Fig. 2) that consist oftrimers of viral gp64 protein (Oomens et at., 1995). The
budded virus form is responsible for cell to cell transmission within an infected insect.
Budded virus is the form used in the laboratory as an expression vector in cultured
insect cells; it is not able to initiate an infection in a host insect (Monsma et at., 1996).
The gp64 protein is responsible for the fusion of the viral envelope and the cell
membrane to initiate infection by AcMNPV (Blissard and Wenz, 1992; Monsma and
Blissard, 1995).
Vit.IEnvelope:
I-i---Nuclcocapsid
Figure 2. Schematic drawing of AcMNPV budded virion.
The polyhedrin protein gene, as mentioned previously, is involved in the formation
of ODV. In the absence of polyhedrin protein budded virus is still secreted by infected
cells, though polyhedra are not formed. As such, this gene is dispensible for propagation
of the virus in cell culture (Miller, 1996). This, coupled with the high levels of
transcription directed by the promoter for this gene provide an opportunity to exploit the
biology of the system for the production of recombinant proteins. Genes placed under
transcriptional control of the polyhedrin (poth) gene promoter can be recombined into
the viral genome to produce a virus stock. The length of the viral capsid is flexible and
is able to accommodate increases in the viral genome length (Fraser, 1986). Upon
infection of an appropriate host cell this virus will direct high level expression of the
desired gene product between 24 to 48 hours postinfection.
Some characteristics of BEVS that can limit its usefulness should be considered.
Infection of cells by rBV can cause changes to the cellular processes which can lead to
differences in expression and modification of proteins as infection proceeds (Kovacs et
at., 1991; Velardo et at., 1993). Different insect cell lines that are used as hosts for rBV
mediated expression can exhibit differences in the processing of over-expressed proteins
(Kost et at., 1997). Furthermore, in some cases, high-level expression can exceed the