MultiBac complexomics

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MultiBac complexomics Expert Rev. Proteomics 9(4), 363–373 (2012)

Simon Trowitzsch1, Dieter Palmberger2, Daniel Fitzgerald3, Yuichiro Takagi4 and Imre Berger*1 European Molecular Biology Laboratory (EMBL) and Unit of Virus Host Cell Interactions (UVHCI), CNRS‑EMBL-UJF UMR 5322, 6 rue Jules Horowitz, F-38042 Grenoble Cedex 9, France 2 Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria 3 Geneva Biotech, 14 Chemin des Aulx, 1228 Plan-les-Ouates Geneva, Switzerland 4 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202-5126, USA *Author for correspondence: Tel.: +33 476 207 061 Fax: +33 476 207 199 [email protected] 1

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Recombinant production of multiprotein complexes is an emerging focus in academic and pharmaceutical research and is expected to play a key role in addressing complex biological questions in health and disease. Here we describe MultiBac, a state-of-the-art eukaryotic expression technology utilizing an engineered baculovirus to infect insect cells. The robust and flexible concept of MultiBac allows for simultaneous expression of multiple proteins in a single cell, which can be used to produce protein complexes and to recapitulate metabolic pathways. The MultiBac system has been set up as an open-access platform technology at the European Molecular Biology Laboratory (EMBL) in Grenoble, France. The performance of this platform and its access modalities to the scientific community are detailed in this article. The MultiBac system has been instrumental for unlocking the function of a number of essential multiprotein complexes and recent examples are discussed. This article presents a novel concept for the customized production of glycosylated protein targets using SweetBac, a modified MultiBac vector system. Finally, this article outlines how MultiBac may further develop in the future to serve applications in both academic and industrial research and development. Keywords: BEVS • glycosylation • mediator • MultiBac • multiprotein complexes • transcription • transnational access/training programs

The MultiBac concept

Mounting evidence supports the view of cells as a collection of multicomponent molecular machines rather than made up of single proteins acting in isolation. These multiprotein molecular machines are thought to carry out key catalytic activities [1,2] . Experiments addressing the interactome, for example, have shown that a given protein interacts on average with five to ten partners in eukaryotes [3,4] . The MultiBac system was specifically developed to enable the production of such eukaryotic multiprotein complexes, which typically could not be purified efficiently from their source or by using existing recombinant technology. In particular, MultiBac was designed to fulfill the following criteria: it should be tailor-made for large eukaryotic multisubunit complexes meaning that the system should be able to accommodate many large genes and allow their concomitant expression; it should assure the isolation of high-quality, homo­geneous products for structural biology and other applications; it should allow for rapid revision of expression experiments; and it should be robust and usable in the hands of non­specialists. Initially, the MultiBac system was mainly employed in academic research. More recently, there has been considerable and increasing demand in 10.1586/EPR.12.32

pharmaceutical research for advanced production technologies such as MultiBac. MultiBac technology

The cornerstone of the MultiBac system is the MultiBac baculoviral genome, which has been engineered for facilitating integration of multiple gene expression cassettes via two independent mechanisms: Cre-recombinase-mediated entry and Tn7 transposition (Figure 1A) . The authors have developed detailed protocols for cloning, virus generation, virus amplification and complex production. All vectors, reagents and chemicals are available at the European Molecular Biology Laboratories (EMBL) in Grenoble, France [5–9] . Essential elements of the MultiBac system are tailor-made, synthetic, custom-designed DNA plasmid modules that, at the smallest feasible size, contain all elements required for efficient gene expression, generation of multigene constructs and integration into the baculoviral genome (Figure 1B) . The system comprises two families of DNA transfer vectors called ‘acceptors’ or ‘donors’. Both acceptors and donors contain gene integration sites flanked by baculoviral late promoters and terminators. Each DNA transfer vector has a different resistance marker, and all donors and acceptors contain a LoxP sequence

© 2012 Expert Reviews Ltd

ISSN 1478-9450

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As a consequence, introduction of isoforms, mutations or truncations into indiGene A vidual proteins can easily be carried out on the gene level. The DNA module with LoxP SpecR the corresponding expression cassette is excised from the multigene construct by LoxP Cre Gene B Gene D adding Cre, followed by manipulating the AmpR excised DNA in isolation, and reintroducF-replicon AmpR ChlR × ing the altered gene into the multigene ­construction via Cre–LoxP fusion. Tn7 Tn7 KanR lacZ The MultiBac system has undergone Gene C continuous improvement since its first mini-attTn7 R introduction. Virus performance and cell Kan viability were significantly improved by Tn7 deleting the viral genes v-Cath and chiA, Expert Rev. Proteomics © Future Science Group (2012) which are responsible for proteolytic and Figure 1. MultiBac methodology. (A) The MultiBac system consists of the MultiBac apoptotic activities imposed by the virus on baculoviral genome, which is maintained as a bacterial artificial chromosome in the host [5] . Protein production considerspecialized Escherichia coli cells. The F-replicon guarantees low copy numbers of the ably benefits from these modifications by bacmid in bacterial cells. Integration of expression cassettes can be realized by Tn7 delayed apoptosis of the host cell (allowing transposition via Tn7 attachment sites that are embedded in a lacZ gene sequence on the for longer expression times) and the prodbacmid, or by Cre-mediated recombination via LoxP imperfect repeat sequences (red circle). (B) For multiprotein production, expression cassettes (e.g., genes A–D) are cloned uct quality is often enhanced by reduced into acceptor and donor vectors and subsequently assembled by Cre-mediated proteolytic breakdown of the target [5] . A recombination via LoxP sequences. Donor vectors contain the R6Kγ conditional origin of drawback of every baculovirus expression replication (purple boxes), whereas acceptor vectors carry standard origins of replication system is the genetic instability of viruses (ColE1; orange box). Multigene transfer vectors are integrated via Tn7 transposition into during amplification and passaging. This the baculoviral genome via Tn7 attachment sites provided by the acceptor vector. Expression cassettes contain the promoter sequences p10 or polH and SV40 or HSVtk genetic instability may become particularly terminator sequences. problematic when very large complexes have to be encoded in the viral genome. for site-specific recombination by Cre-recombinase. Donors and However, specific protocols have been delineated to minimize acceptors differ in their origin of replication. Acceptors have a gene deletions [6] . Also, coinfection strategies with several multistandard origin of replication derived from the common BR322 gene viruses may be taken into consideration for improved proorigin found on many generic plasmids. Donors, in contrast, duction of large protein assemblies. Ultimately, extensive engicarry the conditional R6Kγ origin of replication, which requires neering of the baculoviral genome will contribute to overcome the presence of a pir gene in the bacterial strains used for their viral instability in the future. propagation. Donors cannot replicate in regular cloning strains that do not have the pir gene, unless fused to an acceptor by the MultiBac platform at the EMBL Cre–LoxP reaction. The acceptor then provides the BR322 origin At the EMBL, we have established MultiBac as the central of replication in the acceptor–donor fusion, which can be readily pipeline for the Eukaryotic Expression Facility. The MultiBac propagated in any bacterial strain. platform operates as part of the Partnership for Structural Biology Since each individual donor or acceptor plasmid contains a that unites local French and international institutions including distinct resistance marker, all possible acceptor–donor fusions the European Synchrotron Radiation Facility. Based on our timecan be selected based on the encoded combination of resistance tested protocols, we implemented standard operating procedures markers. This is achieved by challenging bacterial cultures con- for our platform, to achieve maximum throughput and full control taining the fusions with combinations of antibiotics. Due to this of complex assembly and production for nonspecialist users modular concept, genes present on acceptors and donors can be (Figure 2) . The operating process is illustrated in Figure 2A . First, combinatorially assembled into multigene expression constructs recombinant baculoviral genomes (bacmids) that contain the using Cre-meditated recombination in a simple one-step reaction inserted expression constructs are selected by a simple blue–white (Figure 1B) . Single, double and triple expression constructs can be screening step in Escherichia coli and isolated in the form of large generated and selected in a process that is amenable to automation (>130 kb) bacterial artificial chromosomes by applying a standard on a liquid-handling robot [9,10] . The MultiBac system greatly alkaline lysis and isopropanol precipitation protocol. Purified facilitates manipulation and variation of single genes in multi- bacmid DNA is used to transfect insect cells via mixing with gene expression constructs, since each expression cassette can lipidic transfection reagent in multi-well tissue culture plates be specifically isolated from the multigene expression construct containing insect cells spread in monolayers. In these wells, the by making use of the excision activity of the Cre enzyme [9,10] . initial virus (V0) is produced within 48–60 h and harvested in the MultiBac

364

Donor vectors

Acceptor vector

Expert Rev. Proteomics 9(4), (2012)

MultiBac complexomics

media. For qualitative analysis of expression of the target protein or protein complex at the earliest possible stage by western blot, the infected cells are harvested in phosphate buffered saline after

Bacmid selection

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2–3 days from the plate. Typically, a miniaturized small-scale purification reaction can be carried out at this stage, in particular, if affinity tags are attached to the proteins of interest  [7] . The V0

Bacmid isolation

YFP

1

1´

C

2

2´

M

WB

Transfection

Production of V0 YFP

SDS-PAGE

Production of V1

Expression analysis Expert Rev. Proteomics © Future Science Group (2012)

Figure 2. MultiBac platform at the Eukaryotic Expression Facility, European Molecular Biology Laboratory, Grenoble, France. (A) Standard procedure using optimized protocols for protein production at the Eukaryotic Expression Facility (EEF). In brief, transfer plasmids are integrated into the baculoviral genome and positive transformants are selected by blue–white screening. MultiBac bacmids are isolated from bacteria and adhesive Sf21 insect cells are transfected in six-well plate formats. V0 is used to infect Sf21 shaker flask cultures and V1 is harvested. Protein production can be monitored throughout the whole process via fluorescent reporters, SDS-PAGE, and/or western blotting. Proteins or protein complexes can be purified after approximately 2 weeks. Productive virus is stored in the form of baculovirus‑infected insect cell stocks [60] . (B) Five double-deck shakers, (C) two sterile hoods and a light microscope (inset), and (D) a spectrofluorometer are routinely used for protein production and monitoring in the EEF at the European Molecular Biology Laboratory. V0 : Initial virus; V1: Amplified virus; WB: Western blot; YFP: Yellow fluorescent protein. www.expert-reviews.com

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virus is amplified in suspension cultures (25–50 ml in volume) and protein production is confirmed by SDS-PAGE of lysates prepared from identical quantities of cells (106 cells) withdrawn at 12–24 h intervals. A fluorescent marker, yellow fluorescent protein, has been engineered into the baculoviral genome and allows monitoring of virus performance. When the yellow fluorescent protein signal reaches a plateau, recombinant expression is at peak levels [7] . We decided that for our means and purposes the only relevant criterion in a recombinant production experiment is actual protein production, which in turn has to be assessed as soon as possible to continue or abort expression experiments. Consequently, we eliminated virtually all generic quality control steps from the pipeline, notably viral titer determination by plaque assay, which can be particularly time consuming. We based this decision on our previous observations that viruses with strong titers did not necessarily display useful production characteristics [6] . Our resulting operating procedure requires approximately 2 weeks until protein production at quantities sufficient for structural and functional characterization is established. If required, large-scale protein production at any chosen scale can then be carried out by further expansion of production virus following our protocols [6] . Typically, a virus volume of 50 ml is sufficient for most applications in our hands. If larger quantities are required, we prepare virus and store it in the form of frozen aliquots of infected cells. This procedure offers the advantage to exactly reproduce production runs even several years after the initial expression experiment was carried out [6] . The Eukaryotic Expression Facility at EMBL is equipped with two sterile hoods, five double-deck shakers, a light microscope and a spectrofluorometer (Figure 2B) and supports on average 25–30 users in parallel with 50–60 protein production projects. These projects are typically directed toward structural biology including electron microscopy and x‑ray crystallography, or are directed toward the production of high-value biologics, each imposing high demands on the quality of r­ecombinant protein material produced. MultiBac training: transnational access programs of the European Commission

Today, the MultiBac system is already being used in around 450 academic and industrial research laboratories worldwide, for a range of applications. Apart from its successful application in basic research of multiprotein complexes, the MultiBac system has also catalyzed drug discovery and proved to be particularly useful for producing complex biologics including viruslike particles and candidate vaccines for human papilloma virus (HPV) [11,12] . The MultiBac platform at the EMBL offers training and access for scientists who want to learn the technology as part of infrastructure projects (P-CUBE, BioStruct-X) funded by the European Commission (EC) in Framework program 7. The program P-CUBE [101] was installed to offer scientists across Europe, from academia and industry, access to state-of-theart technologies in protein production and high-throughput 366

structural biology, and to disseminate expertise in contemporary structural and mole­cular biology. Within Framework program 7, P-CUBE was the first project to bring together this mix of research, networking and service activities. Leading scientists from the University of Zürich, Switzerland, the University of Oxford, UK and the EMBL now participate in this program. Researchers can join the P-CUBE program via a competitive ‘Transnational Access’ procedure, where an independent panel of scientists selects proposals based on excellence and feasibility as the sole criteria. In P-CUBE, the MultiBac platform at the EMBL was oversubscribed within the first few months of the 4-year program, underscoring the high demand in the research community for powerful as well as user-friendly technologies for the expression of eukaryotic multiprotein complexes. The access activities of the MultiBac platform at EMBL will be continued in BioStruct‑X [102] , a new 4-year EC Framework Program 7 I3 infrastructure program that was launched at the end of 2011. Automated expression of proteins and protein complexes in eukaryotic hosts is of paramount significance in the postgenomic era and is certainly bound to become increasingly sought-after in many fields of biology. Therefore, the P-CUBE and BioStruct‑X programs put special emphasis on the development of auto­ mation for high-throughput sample production, crystallization and structure solution, all of which are the subject of specific joint research activities between the partner laboratories of these I3 consortia. The P-CUBE and BioStruct‑X programs can be expected to set entirely new standards in structural research of eukaryotic multiprotein complexes in the future by optimization of workflows and hardware development. The MultiBac platform technology now being implemented in numerous laboratories in Europe and beyond is expected to play a pivotal role in this vibrant field of research. MultiBac impacts structural biology of protein complexes

In addition to its major impact on the production of previously unavailable multiprotein complexes for basic and applied research, MultiBac also offers obvious advantages for structural biology, in particular for x‑ray crystallography. Often, proteins have to be truncated and low-complexity regions removed to yield specimens that can be successfully crystallized. Crystallization of single proteins, which need to be modified in this manner, is already laborious. For multisubunit complexes, truncations and variations for each subunit can be mandatory, and these variants then need to be combinatorialy assembled. The modular setup of the MultiBac system has addressed this challenge by implementing an efficient robotic approach to combine multiple genes and their variants in a streamlined and parallelized, automatable process termed tandem-recombineering, which can be conveniently performed by a liquid-handling workstation [7,13,14] . Many important complexes have been produced using MultiBac and their structures were solved [15–19] . A selection of multiprotein complexes, which have been produced to date using the MultiBac system, are summarized in Table 1. Recent notable MultiBac exploits are the crystal structures of Expert Rev. Proteomics 9(4), (2012)

MultiBac complexomics

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Table 1. Protein complexes produced by utilizing the MultiBac system. Protein complex

Expression yields (mg/l)

Proteins Subunits

Size (kDa)

Origin

Purification strategy

Ref.

APC

0.5

8 proteins 5 proteins

14–200

Saccharomyces cerevisiae

AC/IEC/SEC

[15]

LKB

10

3 proteins

~37

Homo sapiens

IMAC/IEC/SEC

[16]

Rad–Hus

3

3 proteins

~32

Homo sapiens

IMAC/IEC/SEC

[18]

Glu

NA

2 proteins

~45

Xenopus laevis/Rattus norvegicus

IMAC/SEC

[20]

Chp–Tas

4

2 proteins

10–55

Schizosaccharomyces pombe

AC/IEC/SEC

[21]

Med

15

7 proteins

14–70

Saccharomyces cerevisiae

IMAC/IEC/SEC

[25]

AC: Affinity chromatography; APC: Anaphase-promoting complex; Chp–Tas: Chp1–Tas3 complex; Glu: GluN1/GluN2B N-methyl- d -aspartate receptor; IEC: Ion exchange chromatography; IMAC: Immobilized metal affinity chromatography; LKB: LKB1–STRAD–MO25 complex; Med: Mediator head module; NA: Not available; Rad–Hus: Rad9–Rad1–Hus1 complex; SEC: Size exclusion chromatography.

N-methyl-d-aspartate (NMDA) receptors and the Chp1–Tas3 complex implicated in neurotransmission and RNA interference, respectively (Figure  3A) [20,21] . NMDA receptors were shown to be essential for basic brain development and function and are activated by the binding of glycine and glutamate molecules [22] . Ion channel activity of NMDA receptors is further regulated allosterically by small molecules. Karakas and colleagues determined the crystal structure of the amino-terminal domains of GluN1 and GluN2B of the NMDA receptor bound to ifenprodil and revealed the molecular determinants for phenylethanolamine binding (Figure 3B) [20] . These structural details give mechanistic insight into how small molecules inhibit the activity of NMDA receptors and may help guide the design of new therapeutic candidates for the treatment of neurological disorders [20,23] . An important subassembly of the RNA-induced initiation of transcriptional silencing (RITS) complex was crystallized following production with MultiBac (Figure 3A) . This complex plays a key role in heterochromatin formation at a vital interface of RNA interference mechanisms and genomic organization in eukaryotes [21,24] . The crystal structure reveals the presence of a central platform formed by the RITS complex proteins Chp1 and Tas1, which bridge the chromatin substrate and the RNAi machinery [21] . The study provides evidence that the RITS complex may consist of folded modules that are connected via flexible, unstructured linker regions [21] . MultiBac enables structure determination of the Mediator head module

A highlight of results produced using MultiBac is the crystal structure of the Mediator head module solved recently in the Takagi laboratory [25] . Eukaryotic gene transcription by RNA polymerase II (Pol II) is regulated by intricate machinery comprising a plethora of proteins that are arranged in multiprotein assemblies [26–29] . Determination of the 3D structure of Pol II from Saccharomyces cerevisiae by x-ray crystallography was a hallmark achievement that was acknowledged by the Nobel Prize in Chemistry in 2006 [30–32] . These studies were possible because www.expert-reviews.com

the Kornberg laboratory was able to obtain sufficient quantities of Pol II from yeast. Purification was possible because of the relatively high copy number of endogenous Pol II molecules in yeast. The evolutionary conserved Mediator is another essential component of the eukaryotic gene transcription machinery and regulates Pol II activity [33–35] . Mediator was first discovered in yeast and was shown to be composed of 21 proteins organized in three modules: a head, a middle/arm and a tail [36,37] . The head module was shown to be essential for Mediator f­ unctions [38–42] . In contrast to Pol II, high-resolution structure determination of Mediator and its subcomplexes has lagged far behind due to the intrinsic complexity of the molecule and the very low copy number of Mediator in cells. Initial samples of recombinant Mediator head module were obtained by a coinfection approach using a conventional baculovirus expression system and paved the way for structural studies [41] . However, this approach had distinctly suffered from a lack of reproducibility and lengthy preparation phases of the recombinant baculoviruses [41] . The MultiBac system enabled recombinant production of the complete Mediator head module comprising subunits Med6, Med8, Med11, Med17, Med18, Med20 and Med22 [38] . This complex could be purified to homogeneity, crystallized and its structure subsequently determined by x-ray crystallography (Figure 3C & 3D) [25] . The structure elucidation of the Mediator head is an illustrative example of the utility of the MultiBac system. All genes coding for the seven subunits of Mediator head module were placed on a single recombinant MultiBac baculovirus with optimized protein production properties including reduced protease activities and the yellow fluorescent protein reporter as a marker for virus performance (Figure 3E) [6,25] . The application of MultiBac resulted in superior quality of the recombinant protein complex, hitherto unattainable reproducibility of the expression experiments and proved to be a pre-requisite for successful crystallization and structure determination. Furthermore, the modular nature of MultiBac greatly facilitated the replacement and variation of subunits for optimal crystal growth without de novo ­reconstruction of the multigene expression construct. 367

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Trowitzsch, Palmberger, Fitzgerald, Takagi & Berger

GluN2B Chp1 GluN1b

Tas3 NTD

Med22

Med11 Med17

Fixed jaw Neck

Med20

Med6

Med11

Med6 Med8

Med8

Med18

Movable jaw

Med22

Med17

MultiBac Med17 YFP LoxP AmpR

R6Kg

×

F-Replicon

Med18 Med6 Med8

KanR lacZ mini-attTn7 Med11

Med22

Tn7

Tn7

Med20 Med17

Med20 Med8

Med6 Med18

Med11 Med22 Expert Rev. Proteomics © Future Science Group (2012)

Figure 3. MultiBac enables crystallography of complexes. (A) Crystal structure of Chp1–Tas3 complex [21] . Ribbon diagram showing Chp1 subunit in blue and Tas3 NTD in red. (B) Crystal structure of a GluN1/GluN2B N-methyl- d-aspartate receptor [20] . Ribbon diagram showing the NTDs of GluN2B (green) and GluN1b (blue). The bound ligand, ifenprodil (gold), is shown in stick representation. (C) The structure of the seven-subunit Mediator head module (223 kDa) is shown in a schematic view (top) and in a ribbon representation with experimental electron density in grey around the model (bottom) [25] . (D) A multihelix bundle is central to the Mediator head module and key for its structural integrity. Subunits are color-coded. (E) The composite MultiBac baculovirus with all seven genes (inserted by Tn7 transposition) and a YFP reporter (inserted by Cre-mediated recombination) is depicted. A representative gel section from SDS-PAGE shows the quality of purified Mediator head module (right). NTD: N-terminal domain; YFP: Yellow fluorescent protein.

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The structure of the Mediator head module reveals how multiple proteins can be assembled in a complex to provide stability and flexibility in order to accommodate numerous interactions (Figure 3C) [43] . Notably, the neck of the Mediator head module is formed by a multihelical bundle, in which five of the seven subunits of the module are intertwined in a novel structural arrangement (F igur e   3D) . Furthermore, the complicated, intermolecular arrangement of the subunits explains why previous attempts to reconstitute the Mediator head module from individually purified components failed. The structural analysis of Mediator Head module underscores that such complex assemblies need to be studied as intact entities to understand their physiological functions. Sugar it up: SweetBac

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line with an inducible mammalian-like protein N-glycosylation machinery [56] . However, all of these approaches are based on Sf 9 cells, and for the expression of secreted proteins, cell lines derived from Trichoplusia ni have been demonstrated to produce significantly higher amounts in many cases [57,58] . The thus developed SweetBac system [59] , a platform for the expression of mammalianized proteins in insect cells that is based on MultiBac, might serve to solve the problems stated above. In its first embodiment, a so-called glycomodule, which consists of open reading frames coding for the Caenorhabditis elegans N-acetylglucosaminyltransferase II and the bovine b1,4-galacto­ syltransferase I, controlled by polyhedrin and p10 promoters respectively, was introduced in the LoxP site of the MultiBac baculoviral genome. The resulting SweetBac backbone was subsequently used as a shuttle vector for the expression of human HIV anti-gp41 antibody 3D6 integrated in the standard Tn7 site (Figure 4) . Parallel expression of target genes and glycosyltransferases in several insect cell lines gave an overall expression rate comparable with that of transient expression in mammalian cells. Especially for the recently established Tnao38 cell line, yields of 30 µg/ml cell culture supernatant were reported [59] . The presence of terminal galactose residues on SweetBac expressed antibodies was confirmed by analysing PNGase A released N-glycans with MALDI-TOF-MS. The more complex N-glycan structures had no influence on antibodies target binding ability, but caused a significantly enhanced binding to human Fc γ receptor I (CD64). This indicates an increased ability to exert effector functions such as antibody-dependent c­ ellular cytotoxicity. Next generations of the SweetBac platform could comprise a set of viral backbones containing different glycomodules. Thus, this

Market entry of the human papilloma virus vaccine Cervarix® (GlaxoSmithKline Biologicals, Rixensart, Belgium) has, for the first time, shown the great potential of insect cells for the production of therapeutically active proteins [44] . Today, a variety of baculoviral expression systems and suitable insect cell lines ranging from the widely popular Spodoptera frugiperda Sf9 and Sf21 cell lines, to the Trichoplusia ni cell lines BTI-TN5B1-4 ‘High Five’ and BTI-Tnao38, are available [45–48] . A major limitation regarding the production of therapeutic proteins in these cell lines is the lack of complex type N-glycans, often leading to severely reduced efficacy. N-glycans found on proteins expressed in insect cells are mainly of a high mannose type, or are nonfucosylated and core-fucosylated tri-mannose structures [49] . In the past, several approaches have served to improve N-linked glycosylation in lepidopteran insect cell lines by expressing additional glycosyltransferases. Transient systems were mainly based on coinfecting MultiBac SweetBac insect cells with viruses expressing the target gene together with viruses encoding glycosyltransferases [50,51] . A common Glycomodule problem employing such a setup is the low efficiency of coinfections. Another approach ChlR LoxP × for modifying insects N-linked glycosylaAmpR Glycomodule tion pathways is the stable integration of F-replicon glycosyltransferases open reading frames in the genome of a host cell. In the past KanR lacZ decade, several transgenic insect cell lines have been generated successfully, most of mini-attTn7 mini-attTn7 Expression cassette them based on Sf 9 cells [52–55] . The drawback of this strategy is a possible metabolic GentR KanR overload for the transgenic insect cell line, leading to reduced growth characteristics Tn7 Tn7 and long-term stability as well as reduced Figure 4. SweetBac facilitates production of glycoproteins in insect cells. The yields of recombinantly produced proteins. SweetBac system produce custom glycosylated protein targets is shown. A glycomodule Furthermore, the modified N-glycosylation consisting of the Caenorhabditis elegans N-acetylglucosaminyltransferase II and the patterns might influence the functionality of bovine b1,4-galacto­syltransferase I was integrated into the LoxP site of the MultiBac cellular proteins and have a wider impact on genome resulting in SweetBac. The generated viral backbone was subsequently used the robustness of the system. To overcome for the expression of human HIV anti-gp41 3D6 antibody integrated via the standard Tn7 sites. these problems, a recent study has reported Adapted with permission from [59] . the generation of a new transgenic Sf 9 cell www.expert-reviews.com

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Med2 Med15

Med3

Med16 Med5

biomolecules, for example nucleic acids, to be able to isolate active, multicomponent particles. Five-year view: frontiers of protein production

The intricate interplay of molecules defines cellular function. Protein complexes with Med10 Mediator many subunits are the cornerstone of most Med14 Med19 if not all essential cellular processes. An Med1 understanding of the architecture of a proMed21 Med11 Med22 Med31 tein complex is imperative to decipher its Med7 Med6 function. Addressing this vital task will Med17 require considerable effort and continuous SAGA Med8 development of advanced technologies, in Med18 Spt8 TFIID TAF5 Ada3 RNA Med20 particular for the provision of the comTAF1 Spt7 TAF4 TAF12 plex specimens in the quality and quantity Pol II TAF2 required for structure elucidation, and for TAF5 Ada1 GCN5 TAF9 TAF7 TAF12 the provision of next-generation bio­logics Rbp2 TAF11 TAF3 TAF4 Rbp7 Spt20 Ada2 TAF6 including therapeutic proteins. In this TAF13 TAF9 review we have presented technologies that Rbp5 Rbp6 Rbp1 Spt13 TAF10 TAF8 meet this challenge. The MultiBac technolTAF6 Rbp12 TBP Rbp11 Rbp4 ogy has already accelerated progress in sevTFIIH Rbp10 Rbp3 eral distinct fields of research, and we expect Ccl1 Tfb2 its impact to increase substantially over the Rbp9 TFIIA Rbp8 Tfb3 Tfb1 Kin28 TOA2 coming years. Access to this technology Tfb4 TOA1 for scientists from academia and industry Rad3 Tfa2 Ssl1 Sua7 Tfg3 Ssl2 Tfa1 has been made possible by installing the TFIIB Tfg2 MultiBac platform at the EMBL and securTfg1 TFIIE TFIIF ing competitive funding by the EC, allowing users to acquire expert training. Similar facilities are now being implemented at Figure 5. A eukaryotic complexome. Transcriptional activity of class II genes in eukaryotic cells is brought about by a highly regulated network of multiprotein numerous laboratories across Europe and complexes, including Pol II, Mediator, SAGA and general transcription factors, TFIIA, IIB, beyond, which can be expected to exploit IID, IIE, IIF and IIH. The yeast system is shown, but the mammalian system is even more the MultiBac technology concept for a wide complicated. The Mediator head domain is highlighted. Recombinant production of range of applications. these complexes will be of paramount importance for structural and functional studies The complexity of life in the cell is enorby x-ray crystallography and single particle electron microscopy, and poses one of the major challenges for multiprotein expression systems in the near future. The MultiBac mous. For example, the transcriptional system may contribute significantly to solving these key challenges in contemporary machinery for producing eukaryotic messtructural and molecular biology. senger RNAs contains, in addition to the Pol II: RNA polymerase II; SAGA: Spt–Ada–Gcn5–acetyltransferase complex. genetic DNA template, more than a hunapproach is a promising possibility to express therapeutic proteins dred proteins organized in numerous multisubunit complexes with any desired N-glycosylation pattern in the best ­suitable insect (Figure 5) . We anticipate that technologies such as MultiBac will cell line. prove to be instrumental in the structural and functional dissection of systems as complicated as human gene expression by providExpert commentary ing the multiprotein complexes that perform these functions in Structural genomics initiatives have solved thousands of structures hitherto unattainable quality and quantity. of single proteins and protein domains and have identified numerA number of different industries depend on protein expression ous novel folds. These novel folds have to be further analyzed technologies for product development or production. Advanced in detail by biochemical and biophysical means to understand protein expression technologies are used for manufacturing of thertheir function. Furthermore, proteome-wide interaction stud- apeutic proteins, for production of drug targets, and for creation of ies and mass spectrometric analyses have revealed that proteins multiprotein signaling systems. Some technology platforms, such assemble into complex molecular machines, often harboring other as mammalian cell systems for the manufacturing of humanized biomolecules covalently or noncovalently linked to the proteins. therapeutic antibodies are highly advanced. However, technical Recombinant tools will have to be developed to co-produce these limitations in today’s protein expression technologies still impact Med4

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Med9

Expert Rev. Proteomics 9(4), (2012)

MultiBac complexomics

the development of many other classes of highly sought after products. In the field of influenza vaccines, for example, there is great demand for rapid delivery to market of vaccines that protect against multiple seasonal flu strains or against rapidly emerging flu pandemics. The limitations of available vaccines largely reflect the protein production technologies that are currently used to manufacture them. To exploit the full potential of next-generation vaccine technologies, such as virus-like particle vaccines, further advances in protein ­production ­technologies will be required. Leading technologies will become incorporated into the next generation of biopharmaceutical product pipelines. Similarly, the pharmaceutical industry is moving away from traditional drug targets, such as kinases and G-protein-coupled receptors, to develop new targets in less crowded intellectual property fields. The incorporation of improved protein expression technologies will give producers competitive advantages in the race to establish dominance in new target areas. Finally, the evolution of completely new industries for intelligent protein production technologies, such as industries exploiting synthetic metabolic pathways, and the nutrition, flavor and fragrances industries will be additional battlegrounds for the best protein production technologies over the coming years. We anticipate MultiBac and customized conversions such as SweetBac to be among the leading technologies utilized by both established and emerging industries in the future.

Device Profile

‍Acknowledgements

The authors wish to thank all members of their laboratories, in particular C Bieniossek, F Garzoni and T Imasaki, for their assistance, and R Kornberg and T Richmond for support. Financial & competing interests disclosure

S Trowitzsch is a European Commission (EC) Marie Curie postdoctoral fellow. I Berger acknowledges support from the Agence Nationale de la Recherche (ANR), the French Infrastructure for Integrated Structural Biology Initiative (FRISBI), the Centre National de la Recherche Scientifique (CNRS), the Swiss National Science Foundation (SNSF), the European Molecular Biology Laboratory (EMBL) and the EC through the joint EIPOD program, and the EC projects SPINE2-Complexes and 3D Repertoire (Framework Program 6), as well as INSTRUCT, P-CUBE, BioStruct‑X, 4D-CellFate and ComplexINC (FP7). Y Takagi acknowledges funding from the National Science Foundation (NSF; MCB 0843026), the American Heart Association (AHA; 0735395N) and the Showalter Trust Fund. D Palmberger has been supported by the Austrian Science Fund (FWF): TRP 127-B11. I Berger and D Fitzgerald are authors on patents and patent applications addressing certain aspects of the technologies described here. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest or financial conflict with the subject matter or materials discussed in the m ­ anuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Key issues • Multiprotein production techniques will be crucial in academic and pharmaceutical research in the coming decades. • Structural biology will depend more and more on recombinant protein production techniques to address complex biological questions. • Combined efforts of framework programs and multinational initiatives will be beneficial to set new standards in structural research by optimizing workflows and hardware development. • Successful production of therapeutic proteins requires adaptable, tunable and interchangeable expression systems. heterologous multiprotein complexes. Nat. Biotechnol. 22(12), 1583–1587 (2004).

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