REVIEW Open Access
MultiBac: from protein complex structures
to synthetic viral nanosystems
Martin Pelosse1
, Hannah Crocker1
, Barbara Gorda1
, Paul Lemaire2
, Jens Rauch3
and Imre Berger1*
Abstract
The MultiBac baculovirus/insect cell expression vector
system was conceived as a user-friendly, modular toolkit
for producing multiprotein complexes for structural
biology applications. MultiBac has allowed the structure
and function of many molecular machines to be
elucidated, including previously inaccessible high-value
drug targets. More recently, MultiBac developments
have shifted to customized baculoviral genomes that
are tailored for a range of applications, including
synthesizing artificial proteins by genetic code
expansion. We review some of these developments,
including the ongoing rewiring of the MultiBac
system for mammalian applications, notably CRISPR/
Cas9-mediated gene editing.
Multiprotein complexes are vital cornerstones of most, if
not all, biological processes in cells. To understand the
essential mechanisms of these molecular machines, a detailed
understanding of their architecture and interactions
is required. Several complexes, such as ribosomes,
can be purified from native source material in sufficient
quality and quantity for structural analysis. Other complexes
have low endogenous abundance, impeding their
extraction. Heterogeneity can further complicate protein
complex purification and structure elucidation. In higher
eukaryotes in particular, many complexes can contain
distinct subunit isoforms in variable combinations in different
tissues. Moreover, the subunit composition can be
altered according to cell state. For these complexes, recombinant
production and purification by using heterologous
host cell systems is typically required.
We encountered this challenge over a decade ago,
when working with Tim Richmond in his laboratory at
ETH Zürich on multiprotein complexes that control
* Correspondence: imre.berger@bristol.ac.uk
1
The School of Biochemistry and Bristol Synthetic Biology Centre BrisSynBio,
University of Bristol, Tankard’s Close, Bristol BS8 1TD, UK
Full list of author information is available at the end of the article
transcription initiation in higher eukaryotes, including
humans (reviewed in [1]). Transcription is central to all
biological function, and more than a hundred proteins
contribute to its regulation. In humans, many of these
are organized in multiprotein complexes with ten or
more subunits. Post-translational modifications that may
require authentic recapitulation in heterologous expression
experiments are likewise prevalent. Moreover, subunits
can be very large, with individual molecular
weights exceeding 200 kDa. After substantial trial and
error, we realized that heterologous expression in
Escherichia coli, which dominated recombinant expression
then and still is a preferred choice for many today,
was unsuitable for the task at hand. We then turned our
attention to eukaryotic methods, specifically an expression
vector system relying on a recombinant baculovirus,
to infect insect cell cultures for producing the complexes
in which we had interest.
MultiBac origins
Baculovirus was originally trialed as a pesticide, to control
crop damage caused by Spodoptera frugiperda (the
fall armyworm), with limited success initially, although
more positive results have recently been reported [2–5].
Subsequently, pioneering work, in particular by Max
Summers and colleagues, revealed that baculovirus is
useful for very high-level expression of heterologous genes
in insect cell laboratory culture (reviewed in [5, 6]).
Among the many advantageous features of the baculovirus/insect
cell system, the large DNA cargo capacity was
particularly notable. Baculovirus, in contrast to other viruses
that have a crystalline shell, has a flexible envelope
that can grow with increasing size of the genome packaged
within the baculovirion without adversely affecting
baculovirus function. This particular feature, and the—at
least conceptually—relative ease of manipulating baculovirus
in the laboratory by non-expert users, was exciting
and led us on to the development of MultiBac: a baculovirus/insect
cell system specifically engineered for expressing
functional multiprotein complexes in the quality and
© Berger et al. 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
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Pelosse et al. BMC Biology (2017) 15:99
DOI 10.1186/s12915-017-0447-6
quantity required for high-resolution structural and mechanistic
studies [7–11]. MultiBac went on to be broadly
adopted and used for research in both industry and
academic institutions [12].
The MultiBac system, and its application to protein complex
production, has been described in considerable detail
previously [8–11, 13]. In summary, MultiBac consists of an
engineered baculoviral genome, which is based on the A.
californica multiple nuclear polyhedrosis virus (AcMNPV).
This genome was previously adapted for propagation and
manipulation in bacterial cells in the form of a bacterial
artificial chromosome (BAC) [14, 15]. Starting from this
BAC, we progressively removed functionalities that we
identified experimentally to be detrimental to protein complex
production. For instance, proteolytic and apoptotic
viral factors were eliminated [10]. Baculovirus infection of
insect cells is a lytic process as the virus remodels the host
cell machinery for producing baculovirions at high levels,
resulting ultimately in cell death and lysis. In fact, the highest
level of production of the recombinant protein of interest
coincides with the onset of widespread cell lysis, which
can compromise the protein produced. The introduced
genome alterations resulted in a virus that exhibited
delayed lysis of the insect cells, allowing the production of
recombinant protein complexes at very high levels while
the cells seemingly remained intact [11].
Heterologous genes of interest are inserted into the
MultiBac baculoviral genome by means of transfer plasmids,
which are transformed into so-called DH10MultiBac
cells harboring the BAC (Fig. 1). We prepared an
array of small synthetic plasmid DNA precursor modules
to generate multigene transfer plasmids that were
easily inserted into the MultiBac genome by using tandem
recombineering (TR), which can optionally be done
in high-throughput [13, 16–19]. TR is a method we conceived
to facilitate DNA assembly by iteratively exploiting
sequence and ligation independent cloning (SLIC) to
insert genes of interest into individual plasmid DNA
precursors. These are then concatenated by Cre-LoxPmediated
plasmid fusion and then inserted into the MultiBac
baculoviral genome by transposition, catalyzed by
the Tn7 transposase. The transposase is provided in the
DH10MultiBac cells from a separate helper plasmid coexisting
with the BAC. Although we mostly use SLIC to
manipulate our heterologous genes of interest, our plasmid
modules also contain multiple cloning sites for
MultiBac
virion
VLPvaccines,
protein biologics,
therapeutics
Complex structures
(X-ray, Cryo-EM)
Drug discovery
SBDD, HCS
DNA delivery,
gene therapy
Synthetic biology,
genome optimization
Trans-
fection
MultiBac
Multigene construct
Genes, pomoters, terminators,
expression regulatory elements,
fused polygenic ORFs, …
biGBacUSERTR
Viral LoxP
entry site
Fig. 1. The MultiBac baculovirus expression vector system. MultiBac consists of a baculoviral genome that we engineered for optimal multigene
delivery and protein complex expression (left). The MultiBac genome is propagated as a bacterial artificial chromosome (BAC) in Escherichia coli
cells that supply the Tn7 transposition function from a co-existing helper plasmid. Several methods have been successfully applied to assemble
expression cassettes containing genes of interest and gene regulatory elements. We introduced tandem recombineering (TR), a method that
relies on iterative cycles of sequence and ligation independent cloning (SLIC) coupled to Cre-mediated fusion of plasmid DNA precursor elements
[9, 19]. Other alternatives include the uracil-specific excision reagent (USER) and biGBac methods [20, 21], in addition to conventional restriction/
ligation-based cloning techniques. We engineered a second entry option into the viral backbone, which can accept additional functionalities by
site specific recombination into the viral LoxP site. Composite MultiBac baculoviral DNA containing all DNA elements of interest is extracted. Transfection of
insect cell cultures in small scale yields live MultiBac virions. These can be used for a wide range of applications (right). The viral LoxP site is shown as a circle
filled in red. ORF open reading frame, SBDD structure-based drug design, HCS high-content screening, VLP virus-like particle, Cryo-EM structure determination
by electron cryo-microscopy, X-ray X-ray crystallography, Kan kanamycin resistance marker, Amp ampicillin, LacZ LacZα gene enabling blue/white selection,
mini-attTn7 attachment site for Tn7 transposition. The schematic drawing of the baculovirion was kindly provided by Kari Airenne
Pelosse et al. BMC Biology (2017) 15:99 Page 2 of 10
conventional restriction/ligation cloning. Moreover, others
have provided valuable alternatives (such as uracil-specific
excision reagent (USER) cloning and biGBac) to assemble
large transfer plasmids that contain many genes for integration
into the MultiBac baculoviral genome [20, 21]
(Fig. 1). The USER approach, for instance, is a ligationindependent
cloning method utilizing the incorporation of
uracil in the primers to delimit sticky overhangs generated
by exonuclease treatment; USER facilitates assembly of
multigene expression constructs for producing the APC/C
multiprotein complex [20]. The biGBac method provides
a selection of thoroughly validated primer DNAs for multigene
expression cassette assembly relying on the Gibson
method, and facilitated reconstitution of the kinetochore
complex [21]. Note that TR, USER and biGBac (and other
approaches such as conventional restriction digestion/
ligation) are not mutually exclusive methods to assemble
DNA into multi-expression cassettes for insertion into
MultiBac baculoviral genomes but can be used in combination
according to the requirements of the project.
Accelerating multiprotein complex structural
biology
MultiBac was first adopted by the structural biology
community and has enabled structure elucidation of
many multiprotein complexes, illuminating their cellular
functions. Here, we show a selection of recent structures
obtained with MultiBac-produced specimens [22–31]
(Fig. 2). Noteworthy in this context are the highresolution
structures of influenza polymerase—the longelusive
complex that the influenza virus uses to replicate
and transcribe its genome [32–35]. Influenza polymerase
consists of three subunits, PA, PB1 and PB2, with many
functions, including cap-snatching and endonuclease activities.
Since its discovery decades ago, influenza polymerase
has been studied intensively, but atomic
resolution structures of this coveted drug target have
remained elusive owing to lack of high quality samples.
Influenza polymerase production by using the MultiBac
system allowed the polymerase to be crystallized. The
crystal structures of polymerases from three major influenza
types, A, B and C, were determined, revealing functional
aspects of these protein machines in
unprecedented detail (Fig. 3). Interestingly, efficient production
of influenza A and B polymerases prenecessitated
the implementation of a polyprotein approach
mimicking the strategy certain viruses, such as
coronavirus, exploit to realize their proteome. In our
polyproteins, the genes encoding the subunits of the
complex studied are combined in a single large open
-Mannosidase
Pds5B-
cohesin
complex
IFIT1
Separase-
securin
Scc2
NELF-
AC
Ca2+ Sensing Receptor
Acetyl-CoA
carboxylase
Sphingo-
myelinase
mTOR Complex 1
Raptor
Raptor
Fig. 2. Multiprotein complex structural biology. A selection of recent near-atomic structures of MultiBac-produced specimens, determined by
X-ray crystallography or electron cryo-microscopy [22–31]. In the case of mTOR complex 1, heterologous Raptor was produced by using MultiBac
and crystallized, and the crystal coordinates fitted into a cryo-EM map of endogenously purified holo-complex in a hybrid approach [31]. Accession
numbers from top left to bottom right are EMDB 8166 (α-mannosidase), PDB 5K5S (Ca2+
sensing receptor), PDB 5U1T (separase-securin complex), PDB
5FIB (sphingomyelinase), PDB 5L3X (NELF-AC), PDB 5I6I (acetyl-CoA carboxylase), PDB 5UDL (IFIT1), PDB 5ME3 (Scc2), EMDB 4029 (Pds5B-cohesin complex)
and PDB 5FLC (mTOR complex 1)
Pelosse et al. BMC Biology (2017) 15:99 Page 3 of 10
reading frame (ORF). This ORF is integrated into the
MultiBac genome, giving rise to a large protein that
is subsequently processed by a highly specific protease
(in our case TEV NIa) that is likewise encoded by the
ORF. The protease cleaves specific recognition sites
present between the subunits, thus releasing them in
a precise stoichiometric ratio, resulting in the functional
complex [8, 36, 37].
MultiBac evolution: synthetic viral nanosystems
When MultiBac was initially conceptualized, the DNA sequence
LoxP was engineered into the viral backbone. This
provided the means to integrate further genes of interest
by Cre-LoxP fusion into this second locus, distal to the
Tn7 attachment site. Our incentive was to provide the option
for distributing the genes encoding a multiprotein
complex, in case it became inconvenient to integrate
many expression cassettes into a single site on the viral
backbone. This turned out to be less of a concern than we
had anticipated, and therefore this viral LoxP site was initially
used only to integrate a fluorescent marker, yellow
fluorescent protein (YFP). This allowed real-time virus
performance to be tracked and the optimal timepoint for
harvesting the cell cultures to be determined [13]. In
addition, the viral LoxP site turned out to be useful for
introducing specific functionalities, giving rise to customized
genomes with tailored functions (Fig. 4), which we
termed synthetic viral nanosystems (SVNs).
With the Grabherr group in Vienna, we created SweetBac,
a SVN designed to tailor the glycosylation pattern of
secreted proteins expressed in insect cells [38–41]. By
exploiting the viral LoxP site, mammalian glycosylases
were integrated into the BAC, which converted the insectcell-specific
high-mannose type glycosylation into the
complex sugars associated with human proteins. Conversely,
by introducing the deglycosylases PNGase or
EndoH, respectively, we created SVNs that entirely
removed sugars from the recombinant proteins. This is
often preferred for structure elucidation by X-ray crystallography,
in which a highly homogenous sample is required,
ideally lacking variable extensions, such as sugars,
which could impede crystallization. A further posttranslational
modification that can lead to heterogeneity is
phosphorylation, which may or may not be authentic in
insect cell expression. In our study of Isw, a chromatin remodeling
enzyme complex, we found that the SVN that
expresses λ-phosphatase from the viral LoxP site could remove
the phosphate groups quantitatively from the recombinant
complex, resulting in a highly homogenous
sample that could be dissected mechanistically [42, 43].
Following the same logic, further SVNs were developed
for improved manufacturing of selected classes of
protein biologics of pharmacological interest. Kinases
are cornerstones of signal transduction in cells, and
many disease states, notably cancer, are caused by malfunction
of kinases and their interactions. Smallmolecule
drugs acting as kinase inhibitors are prolifically
used in cancer chemotherapy, and the discovery of new
and better kinase inhibitors is a major driving force in
oncology research and development. A vital prerequisite
to sustain these efforts is the supply of high-quality recombinant
kinases for screening and structure-based design
approaches. Kinases are often inherently fragile
proteins that require helper molecules, such as chaperones,
for proper folding during expression. To enhance
recombinant production of properly folded and soluble
kinases, we designed SVNs that contain up to seven
chaperones in the viral LoxP site (Fig. 4). To balance the
quantities of individual chaperones produced, we again
applied our polyprotein strategy. The resulting “kinase
factory” produced previously unobtainable kinases at
high levels, in soluble and functional form.
Another class of protein biologics of acute interest are
virus-like particles (VLPs), which mimic live viruses but
FluA FluB FluC
*
Fig. 3. Flu polymerase structures. Crystal structures of MultiBac-produced polymerases of the three major influenza subtypes, fluA, fluB and fluC,
have been determined [32–35]. For fluA and fluB, a polyprotein approach was used to balance stoichiometry. Subunits are expressed as a single
open reading frame (ORF) encoding a large polyprotein, which is then processed by highly specific protease included in the ORF [18, 37]. By contrast,
fluC was crystalized using conventional co-expression of the subunits. FluA and fluB polymerase in the crystals are bound to cognate RNA; the fluC
structure represents the apo form of the complex. More recently, the molecular interactions between fluA polymerase and a short C-terminal domain
(CTD) peptide from RNA polymerase II (Pol II) were elucidated [34] (left). The asterisk denotes the position of Pol II CTD peptides bound. PDB accession
numbers are: fluA polymerase, PDB 5M3H; fluB polymerase, PDB 4WSA; fluC polymerase, PDB 5D9A
Pelosse et al. BMC Biology (2017) 15:99 Page 4 of 10
are safe and non-infectious as they lack genetic content.
VLPs therefore represent promising candidates for vaccination
to combat infectious diseases [44–46]. The influenza
M1 capsid is a powerful driver of VLP formation in insect
cells. We therefore integrated a gene that expressed M1
from influenza strain H1N1 into the viral LoxP site, together
with a gene encoding the fluorescent protein
mCherry, which we used to monitor virus performance
(Fig. 4). We used this SVN for high-throughput production
and validation of an array of influenza VLPs with a
large number of immune-modulating mutations in their
hemagglutinin (HA) gene [47].
An unusual application of MultiBac technology
involved the generation of recombinant adenovirus associated
vector (rAAV) for gene therapy [48–51]. Here,
MultiBac was used to provide all components (REP78,
REP72, AAV virion coat proteins and the transgenes)
required to co-produce gene-therapy-competent rAAVs
in Sf9 insect cell cultures. The components spontaneously
assemble to form intact rAAVs. The system was
used to produce rAAVs encoding leptin, for gene therapy
of obesity in laboratory rodents. Although the gene therapy
successfully resulted in weight-loss, it was nonetheless
noted in the report that best results were achieved
when the gene therapy regimen was accompanied with
exercise using a tread-mill [48], somewhat dampening
optimism that exercise-free weight loss may be achievable.
Originally, this approach involved co-transfection
of three individual MultiBac viruses to obtain intact
rAAVs, constraining logistics. More recent refinement of
the system, including integrating the encoding genes directly
into the genome of the host Sf9 cells, resulting in a
streamlined manufacturing process representing a viable
alternative for rAAV production [48–51].
MultiBacTAG: extending the scope of genetic
code expansion
Genetic code expansion (GCE) incorporates artificial
amino acids into polypeptide chains to create synthetic
proteins with novel functions; it has many applications,
ranging from discovery science to molecular medicine
[52–54]. In GCE, an orthogonal tRNA/cognate synthetase
pair is used to suppress a rare stop codon
introduced at a specific site in a gene of interest. The
tRNA acts as a stop codon suppressor, effectively repurposing
the rare stop codon of choice into a functional
sense codon during protein synthesis, which leads to the
site-specific incorporation of an artificial amino acid
supplemented in the culture medium. A wide range of
functionalities from protein labeling to photo-control
MultiBac
Target gene(s)
SVNs
Kinase
factory VLP factory MultiBacTAG
100
75
37
50
T S ST
*
Flu H1N1 M1
mCherry
M. Mazei PylRS
U6Sf21/ tRNAPyl
Hop, HSP40, P23
PPID, AHSA1
CDC37, HSPA5
TAF13
TBP
TAF11
*
TAF13
uv- +
UAG
ncAA
tRNAPYL
a b c
MultiBac
Ribosome
Fig. 4. Synthetic viral nanosystems. Synthetic viral nanosystems (SVNs) are created by customizing MultiBac baculoviral genomes through integration
of additional functionalities into the viral LoxP site. Selected examples are depicted in the box on the right (panels a–c). a TheMultiBac-based “kinase
factory” contains seven chaperones that assist in the folding of kinase enzymes, thus enhancing the stability and yield of functional protein.
Coomassie-stained SDS-PAGE sections show test expression of KRS1, with the MultiBac virus (left lanes) and the kinase factory (right lanes), respectively.
T total cell extract, S soluble protein. The asterisk denotes KRS1, which was expressed as a glutathion-S-transferase (GST) fusion protein. Molecular
weight marker band sizes (in kDa) are indicated by numbers. The kinase is barely visible in the MultiBac expressions. By contrast, high-level expression
of kinase is observed with the kinase factory. b “Virus-like particle (VLP) factory” was constructed by integrating the gene encoding influenza H1N1 M1
capsid protein into the viral LoxP site, together with the florescent protein mCherry-encoding gene to track virus performance. The VLP factory efficiently
produces influenza virus-like particles decorated with envelope proteins co-expressed from the viral Tn7 site. VLP is shown in a schematic drawing (courtesy
of D. Jordan), with the capsid formed by M1 protein colored in brown. The VLP is devoid of genetic content but displays hemagglutinin (blue spikes) and
neuraminidase (purple spikes) on its surface in a live-virus-like fashion, embedded in a host-cell derived lipid bilayer envelope. An EM image of influenza VLP
produced by using VLP factory is shown beneath the schematic. c The MultiBacTAG SVN. Orthogonal pyrolysyl tRNA (tRNAPyl
) and cognate synthetase
(PylRS) from Methanosarcina mazei inserted in the viral LoxP site enable integration of artificial (non-canonical) amino acids (ncAA) by UAG stop-codon
suppression. The growing polypeptide chain contains the artificial amino acid (ncAA, red star) at the position determined by the UAG codon. MultiBacTAG
was used to integrate a UV-activatable amino acid into a protein complex formed by human transcription factors TAF11 and TAF13, revealing specific
crosslinks in the presence of TBP (drawn schematically in gray). A section from a western blot stained with anti-TAF13 antibody shows the cross-link upon
UV radiation. Reprinted by permission from Macmillan Publishers Ltd: Nature Methods 13:997-1000, copyright 2016 [58]
Pelosse et al. BMC Biology (2017) 15:99 Page 5 of 10
can be introduced by using GCE in biotechnology and
biomedical research [55–57]. Until recently, this method
has been mostly confined to small individual proteins,
expressed in E. coli or mammalian cells, representing a
limited repertoire of cellular activity. Together with the
Lemke and Braese groups, we extended the scope of this
method by combining GCE with the MultiBac system.
This gave rise to MultiBacTAG (Fig. 4), a protein engineering
platform that enables GCE in complex multiprotein
machines expressed in insect cells [58].
To customize the MultiBac baculovirus for GCE, the
pyrolysyl tRNA/tRNA synthetase (PylRS/tRNAPyl
) pair
from Methanosarcina mazei was chosen. The gene encoding
the synthetase and a cassette for producing tRNAPyl
was inserted into the viral LoxP site. The design of the
tRNA production cassette proved to be surprisingly complicated.
The available insect RNA polymerase IIIdependent
promoters for RNA production (Bombyx mori
U6 and Drosophila melanogaster U6) did not function in
the S. frugiperda Sf21 cells that we typically use for
MultiBac-based protein production. Overcoming this
bottleneck required sequencing the Sf21 genome to identify
a Sf21 U6 promoter which supported efficiently the
production of M. mazei tRNAPyl
, leading to effective rare
stop codon suppression and incorporation of artificial
amino acids derived from pyrolysine into the proteins
expressed with the MultiBacTAG SVN.
Current MultiBacTAG applications include artificial
amino acid cross-linking to map interactions in protein
complexes, fluorescence labeling of specific targets to
measure structure and dynamics in proteins and glycolengineering
proteins compatible with human tissue studies.
We anticipate that the platform will also be adopted
for custom-design proteins for therapeutic biotechnology
and pharmaceutical applications. As an example, we used
MultiBacTAG to engineer trastuzumab (also known as
Herceptin), an antibody that associates with cancer cells,
to recognize breast cancer cells in human tissue [58, 59].
MultiBac-based genome engineering by CRISPR/
Cas9
Multigene delivery and subsequent cellular expression is
a key technology for a wide range of applications in biology,
not only in structural research but also cellular reprogramming
and functional pharmaceutical screening.
The construction of multigene circuits in mammalian
cells is a core concept in synthetic biology and requires
efficient delivery of complex heterologous DNA. For certain
cell types, including the widely used HEK293 and
HeLa cells, this can be achieved by plasmid-based transfection.
However, many cell lines, including primary
cells, are recalcitrant to plasmid transfection and therefore
require a different approach. Baculovirus in its original
form is highly selective for infecting insect cells,
which is among the reasons why baculovirus/insect cell
expression can be carried out at standard laboratory biological
safety levels. At very high concentrations, however,
baculovirus can also penetrate mammalian cells.
This process is called transduction rather than infection
as the baculovirus, in contrast to mammalian viral pathogens,
will not replicate in mammalian cells. If the baculoviral
genome that has been transduced into the
mammalian host cell contains an expression cassette
with a mammalian cell active promoter, expression of
the transgene occurs [60, 61]. This “BacMam” gene delivery
approach is attractive owing to the very large
DNA cargo capacity of baculovirus. In principle, a single
baculovirus can deliver many genes of interest at the
same time, a result that is difficult to achieve by
plasmid-based co-transfection.
We customized the MultiBac baculovirus by integrating
the gene encoding vesicular stomatitis virus G protein
(VSV-G), a protein which was shown to substantially increase
the transduction efficacy [62]. The resulting MultiBacMam
baculovirion displays VSV-G glycoproteins on the
virion surface, and was well suited for transducing a wide
range of established mammalian cells with unprecedented
efficacy [63]. Primary cells are a central focus of current
biological research efforts, and multigene delivery in primary
cells is highly desirable; however, suitable tools have
been markedly lacking. We demonstrated that MultiBacMam
potently transduced primary cells (Fig. 5). Moreover,
MultiBacMam could be used to reprogram fibroblasts into
neurons, with virtually identical efficiency as a lentiviral approach.
Of note, MultiBacMam transduction is transient in
nature and does not permanently alter the genome, in contrast
to the genomic integration of lentivirus.
The very large DNA cargo capacity of the baculovirion,
and its aptitude to transduce mammalian cells,
raises the attractive possibility to use this tool, in conjunction
with powerful gene editing technologies, to
rectify genetic aberrations, possibly in whole organisms.
The gene encoding Cas9 and the modalities to produce
guide RNAs and a gene encoding eGFP as a model DNA
cargo were integrated into the MultiBacMam system.
We showed that this MultiBacMam genome engineering
tool could be used for CRISPR/Cas9-mediated insertion
of eGFP into the HMG locus of a range of cell types, including
neurons [63]. Thus, MultiBacMam can now be
developed into a bona fide genome-engineering tool,
characterized by an unsurpassed DNA cargo capacity
that is orders of magnitude larger than the commonly
used gene therapy viral vectors (lentivirus, AAV and
adenovirus) that currently dominate the field.
MultiBac Redux: SynBac
The original AcMNPV genome from which the MultiBac
is derived contains all the information required for the
Pelosse et al. BMC Biology (2017) 15:99 Page 6 of 10
virus to sustain its life cycle in nature, starting from uptake
by the host (the fall armyworm), the infection of
midgut cells, followed by massive amplification and
release of occluded viral particles when the worm lyses.
Many of these functions are dispensable, or even detrimental,
in laboratory cell culture. The advent of powerful
DNA synthesis and assembly technologies has now
made it feasible to “rewire” entire genomes. Genome
improvement by eliminating undesired functionalities is
a major ambition in current synthetic biology. The baculoviral
genome is around 140 kb, which is the size of an
average BAC, and within the size that is capable of
complete de novo synthesis, as recently shown [64].
A disadvantage of currently available recombinant
baculoviral systems, including MultiBac, is genome
instability during virus amplification. The baculovirus
progressively eliminates DNA from its genome when
serially passaged over several generations, often most
severely affecting the heterologous DNA to be expressed
[65–67]. In extreme cases, only a few full-size baculoviral
genomes are present after several rounds of amplification
in cell culture, and small circular DNAs
containing tiny fractions of the baculoviral genome dominate.
These survive by scavenging on the packaging
proteins produced from the full-size specimens, overtaking
the viral population. For academic research requiring
typically only a few milligrams of protein sample, this
can be controlled in a relatively straightforward manner
by avoiding virus overamplification [9, 10, 68]. However,
this is much more difficult and often impossible to
achieve at scales relevant for pharmaceutical manufacturing.
Here, large volumes of virus and therefore many
cycles of amplification are required to infect fermenterscale
production runs. An improved baculoviral genome
resolving this handicap would thus be a major advance.
We carried out a comparative study of all available
baculoviral genomes in sequence data bases and
combined this with data mining to delineate a putative
minimal baculoviral genome, SynBac, that would be capable
of sustaining its propagation and high-level heterologous
protein production in cell culture [69]. Based on
our data, the baculoviral genome was divided into
segments to be individually streamlined through the
elimination of DNA elements that we identified as
dispensable for our purposes (Fig. 6). The maximally
condensed synthetic DNA sections can then be subsequently
grafted back into the MultiBac baculoviral genome
to replace the wild-type sequence, and tested for
MultiBacMam
CRISPR/Cas9iPSneuronHUVEC
Fig. 5. Mammalian multigene delivery and gene editing. The tropism of the MultiBac baculovirus was expanded by pseudotyping. A gene encoding
for vesicular stomatitis virus glycoprotein (VSV-G) was integrated into the viral LoxP site, accompanied by a mCherry gene for tracking
virus performance, resulting in the MultiBacMam virus (bottom right). MultiBacMam efficiently transduces mammalian cell types known to
be recalcitrant to plasmid-based transfection, such as human umbilical vein endothelial cells (HUVECs), neurons and induced pluripotent
stem (iPS) cells, with multigene DNA cargos encoding multiple fluorescently labeled proteins (top three rows). Transduction with MultiBacMam virus
comprising the elements required for CRISPR/Cas9-mediated gene editing and a GFP-encoding gene as model DNA cargo resulted in successful
integration into the HMG locus (bottom row) [63]
Pelosse et al. BMC Biology (2017) 15:99 Page 7 of 10
functionality. Synthetic sections that pass rigorous validation
are then iteratively combined until the minimal,
fully rewired SynBac baculoviral genome is accomplished.
We showed that 30 kb of the MultiBac baculoviral
genome could be altered in this way. The firstgeneration
virus generated (SynBac1.0) was not only
fully functional in terms of high-level protein production,
but already showed a major improvement in genome
stability during serial passaging of the virus (Fig. 6).
This validated the approach and addressed one of the
major current shortcomings of baculoviral expression,
genome instability.
Conclusion and outlook
The MultiBac system was shown in its first configuration
in 2004 [11], which had been developed to address
the challenge of efficient multiprotein complex production
for structural biology applications. Subsequently, we
received requests from laboratories in both academia
and industry exemplifying the need for such a tool-kit, a
trend that has not since subsided. The numerous important
structures elucidated using MultiBac-produced
samples evidenced how useful the system is. We, and
others, invested considerable effort over the years to
streamline MultiBac, rendering it more user-friendly and
accessible, with improvements such as alternative
methods for inserting genes of interest.
More recently, the scope of the system has been expanded
by customizing the genome for specific applications,
as well as altering the tropism of the virus to
enable efficient multigene transfer in a wide range of
mammalian cell types and organisms. In our view, genomic
intervention mediated by nucleases (e.g. Cas9,
TALENs and Zinc-fingers), is a particularly promising
application. Combining gene editing with the very large
(>100 kb) heterologous DNA cargo capacity of baculovirus
could be instrumental for future gene therapy applications.
We anticipate that next-generation genomic
intervention strategies will critically depend on providing
complex multicomponent functionalities, including targeting
activities and host immune-system modulators in
a single multifunctional DNA delivery tool. SynBac, the
synthetic baculovirus, could be a tool of choice,
providing currently unmatched DNA cargo capacity in
an optimized genome.
Acknowledgements
We thank all members of the Berger laboratory, in particular Deepak Balaji
Govinda Raj, for helpful discussions. Kapil Gupta is acknowledged for help
with structure representations. Kari Airenne (University of Eastern Finland)
kindly provided the schematic drawing of the bacuvirion. We thank Daniel
Fitzgerald (Geneva Biotech, Switzerland) and Ismail Moarefi (Crelux GmbH,
Germany) for useful comments on the manuscript, and Tim Richmond (ETH
Zurich, Switzerland) for encouragement and support at the early stages of
the project. This work was supported by the Science Foundation Ireland
(grant number 14/IA/3295, to JR) and the European Commission Framework
Programme 7 projects SynSignal (contract number 613879, to JR and IB) and
ComplexINC (contract number 279039, to IB). The authors acknowledge
Frag. I
II
IV
V
VI
VII
III
150
100
75
50
37
25
T S
MultiBac
150
100
75
50
37
25
T S
SynBac1.0a b
c
MultiBacSynBac1.0
Fig. 6. Minimizing and optimizing the baculoviral genome to create SynBac. a The DNA elements in the wild-type AcMNPV baculoviral genome
were classified according to perceived importance for cell culture applications, derived from exhaustive comparative genome analysis and data
mining [69], and distributed into fragments (I to VII) for rewiring and condensing, with the aim to eliminate surplus DNA and improve virus performance in
the laboratory and in manufacturing. b Experiments with SynBac1.0, a MultiBac-derivative comprising a condensed Fragment I, showed powerful
expression of a test protein (marked by arrowhead), indistinguishable from MultiBac based on Coomassie-stained SDS-PAGE. Numbers indicate molecular
weight marker sizes (in kDa). T total cell extract, S soluble protein. c Serial passaging of SynBac1.0 expressing a large polygenic test open reading frame
(ORF) containing a red fluorescent protein marker (dsRED) evidenced a remarkable increase of SynBac1.0 genome stability compared with MultiBac. More
than 70% of infected cells still produce dsRED in fluorescent micrographs, indicating the presence of intact genomes. By contrast, identically overamplified
MultiBac virus frequently lost dsRED, resulting in a much lower proportion (<20%) of infected cells producing dsRED
Pelosse et al. BMC Biology (2017) 15:99 Page 8 of 10
support by BrisSynBio, a BBSRC/EPSRC funded Centre for synthetic biology
at the University of Bristol (BB/L01386X/1).
Authors’ contributions
MP and IB wrote the manuscript together with input from HC, BG, PL and JR.
All authors have read and agreed the content.
Competing interests
The authors declare competing financial interest. IB is inventor on international
patents (US 61/762.607, EP2403940, EP1723246, EP1945773) covering parts of
the MultiBac technology here described.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published
maps and institutional affiliations.
Author details
1
The School of Biochemistry and Bristol Synthetic Biology Centre BrisSynBio,
University of Bristol, Tankard’s Close, Bristol BS8 1TD, UK. 2
Geneva Biotech
SARL, Avenue de la Roseraie 64, 1205 Genève, Switzerland. 3
Systems Biology
Ireland, University College Dublin, Belfield Dublin 4, Republic of Ireland.
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