the-multibac-protein-complex-production-platform-at-the-embl

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

EMBL Grenoble Outstation and Unit of Virus Host Cell Interactions (UVHCI) UMR5322

Most eukaryotic proteins exist as large multicomponent assemblies with many subunits, which act in concert to catalyze specific cellular activities. Many of these molecular machines are only present in low amounts in their native hosts, which impede purification from source material. Unraveling their structure and function at high resolution will often depend on heterologous overproduction. Recombinant expression of multiprotein complexes for structural studies can entail considerable, sometimes inhibitory, investment in both labor and materials, in particular if altering and diversifying of the individual subunits are necessary for successful structure determination. Our laboratory has addressed this challenge by developing technologies that streamline the complex production and diversification process. Here, we review several of these developments for recombinant multiprotein complex production using the MultiBac baculovirus/insect cell expression system which we created. We also addressed parallelization and automation of gene assembly for multiprotein complex expression by developing robotic routines for multigene vector generation. In this contribution, we focus on several improvements of baculovirus expression system performance which we introduced: the modifications of the transfer plasmids, the methods for generation of composite multigene baculoviral DNA, and the simplified and standardized expression procedures which we delineated using our MultiBac system.

New baculovirus expression tools for recombinant protein complex production.

Prot&eacute;ines eucaryotes plus existent en tant que de grandes assembl&eacute;es multicomposants avec plusieurs sous-unit&eacute;s, qui agissent de concert afin de catalyser les activit&eacute;s cellulaires sp&eacute;cifiques. Beaucoup de ces machines mol&eacute;culaires sont uniquement pr&eacute;sents en faible quantit&eacute; chez leurs h&ocirc;tes indig&egrave;nes, qui font obstacle &agrave; la purification de la mati&egrave;re d&#39;origine. D&eacute;m&ecirc;ler leur structure et le fonctionnement &agrave; haute r&eacute;solution d&eacute;pendra souvent de surproduction h&eacute;t&eacute;rologue. Recombinante expression des complexes multiprot&eacute;iques pour &eacute;tudes structurales peut entra&icirc;ner un investissement consid&eacute;rable, parfois inhibiteur, en main-d&#39;&oelig;uvre et mat&eacute;riaux, en particulier si modifier et diversifier des sous-unit&eacute;s individuelles sont n&eacute;cessaires pour d&eacute;terminer la structure succ&egrave;s. Notre laboratoire a abord&eacute; ce d&eacute;fi en d&eacute;veloppant des technologies qui simplifient le processus complexe de production et de la diversification. Ici, nous passons en revue plusieurs de ces d&eacute;veloppements pour la production de complexe multiprot&eacute;iques recombinante en utilisant le syst&egrave;me d&#39;expression baculovirus/insecte cellule MultiBac que nous avons cr&eacute;&eacute;. Nous avons abord&eacute; aussi parall&eacute;lisation et automatisation de l&#39;Assembl&eacute;e de g&egrave;ne pour l&#39;expression de complexe multiprot&eacute;iques en d&eacute;veloppant des routines robotis&eacute;s pour la g&eacute;n&eacute;ration de vector multig&eacute;nique. Dans cette contribution, nous nous concentrons sur plusieurs am&eacute;liorations des performances du syst&egrave;me baculovirus expression que nous avons introduit : les modifications des plasmides de transfert, les m&eacute;thodes pour la g&eacute;n&eacute;ration de composite multig&eacute;nique baculoviral ADN et les proc&eacute;dures d&#39;expression simplifi&eacute;e et standardis&eacute;e qui nous d&eacute;limit&eacute;es &agrave; l&#39;aide de notre syst&egrave;me de MultiBac.

Nouveaux outils d&#39;expression baculovirus pour la production de complexes de prot&eacute;ines recombinantes.

Proteine eucariotiche pi&ugrave; esistano come grandi assiemi multicomponenti con molte subunit&agrave;, che agiscono in concerto a catalizzare specifiche attivit&agrave; cellulari. Molte di queste macchine molecolari sono solo presenti in basse quantit&agrave; nei loro eserciti nativi, che ostacolano la purificazione da materiale di origine. Disfare la loro struttura e funzione ad alta risoluzione spesso dipender&agrave; sovrapproduzione eterologa. Espressione recombinant di complessi multiproteici per gli studi strutturali pu&ograve; comportare un investimento considerevole, a volte inibente, in manodopera e materiali, in particolare se alterando e diversificazione delle singole subunit&agrave; sono necessarie per la determinazione della struttura di successo. Il nostro laboratorio ha affrontato questa sfida con lo sviluppo di tecnologie che ottimizzano il complesso processo di produzione e diversificazione. Qui, passiamo in rassegna alcuni di questi sviluppi per la produzione di complessi multiproteici ricombinante utilizzando il sistema di espressione di baculovirus/insect cell MultiBac che abbiamo creato. Abbiamo affrontato anche parallelizzazione e automazione dell&#39;Assemblea gene per l&#39;espressione di complessi multiproteici sviluppando le routine di robotiche per la generazione di multigene vettoriale. In questo contributo, ci concentriamo su diversi miglioramenti di prestazioni del sistema baculovirus espressione che abbiamo introdotto: le modifiche di plasmidi i trasferimento, i metodi per la generazione di compositi multigene baculoviral del DNA e le procedure di espressione semplificata e standardizzata che abbiamo delineati utilizzando il nostro sistema MultiBac.

Nuovi strumenti di espressione di baculovirus per la produzione di complessi della proteina ricombinante.

We are witnessing tremendous advances in our understanding of the organization of life. Complete genomes are being deciphered with ever increasing speed and accuracy, thereby setting the stage for addressing the entire gene product repertoire of cells, towards understanding whole biological systems. Advances in bioinformatics and mass spectrometric techniques have revealed the multitude of interactions present in the proteome. Multiprotein complexes are emerging as a paramount cornerstone of biological activity, as many proteins appear to participate, stably or transiently, in large multisubunit assemblies. Analysis of the architecture of these assemblies and their manifold interactions is imperative for understanding their function at the molecular level. Structural genomics efforts have fostered the development of many technologies towards achieving the throughput required for studying system-wide single proteins and small interaction motifs at high resolution. The present shift in focus towards large multiprotein complexes, in particular in eukaryotes, now calls for a likewise concerted effort to develop and provide new technologies that are urgently required to produce in quality and quantity the plethora of multiprotein assemblies that form the complexome, and to routinely study their structure and function at the molecular level. Current efforts towards this objective are summarized and reviewed in this contribution.

Getting a grip on complexes.

Nous assistons &agrave; des progr&egrave;s consid&eacute;rables r&eacute;alis&eacute;s dans la compr&eacute;hension de l&#39;organisation de la vie. G&eacute;nomes complets sont &ecirc;tre d&eacute;chiffr&eacute;s avec toujours plus de rapidit&eacute; et pr&eacute;cision, d&eacute;clenchant ainsi le terrain pour s&#39;attaquer au r&eacute;pertoire de produit de g&egrave;ne entier des cellules, vers la compr&eacute;hension des syst&egrave;mes biologiques enti&egrave;res. Progr&egrave;s de la bio-informatique et les techniques de spectrom&eacute;trie de masse ont mis en &eacute;vidence la multitude d&#39;interactions pr&eacute;sentes dans le prot&eacute;ome. Complexes multiprot&eacute;iques sont pr&eacute;sentent comme des pierres angulaires primordiale de l&#39;activit&eacute; biologique, comme de nombreuses prot&eacute;ines semblent participer, stablement ou transitoirement, aux grandes sous-unit&eacute;s assembl&eacute;es. Analyse de l&#39;architecture de ces assembl&eacute;es et leurs interactions multiples est imp&eacute;ratif pour comprendre leur fonction au niveau mol&eacute;culaire. Les efforts de g&eacute;nomique structurale ont favoris&eacute; le d&eacute;veloppement de nombreuses technologies pour atteindre le d&eacute;bit n&eacute;cessaire pour l&#39;&eacute;tude des prot&eacute;ines simples-l&#39;&eacute;chelle du syst&egrave;me et des motifs de petite interaction &agrave; haute r&eacute;solution. Le glissement actuel vers les grands complexes multiprot&eacute;iques, en particulier chez les eucaryotes, maintenant appelle &agrave; un effort concert&eacute; de m&ecirc;me de d&eacute;velopper et de fournir de nouvelles technologies qui sont requis de toute urgence pour produire en qualit&eacute; et en quantit&eacute; la pl&eacute;thore d&#39;assemblys multiprot&eacute;iques que forment le complexome et d&#39;&eacute;tudier syst&eacute;matiquement leur structure et leur fonction au niveau mol&eacute;culaire. Les efforts actuels vers cet objectif sont r&eacute;sum&eacute;s et examin&eacute;s dans cette contribution.

Obtenir une poign&eacute;e sur les complexes.

Stiamo assistendo a enormi progressi nella nostra comprensione dell&#39;organizzazione della vita. Genomi completi sono essere decifrati con sempre maggiore velocit&agrave; e precisione, quindi ponendo le basi per affrontare il repertorio di prodotto intero gene delle cellule, alla comprensione di sistemi biologici di tutto. Advances in bioinformatica e tecniche di spettrometrihe di massa hanno rivelato la moltitudine di interazioni presenti nel proteoma. Complessi multiproteici stanno emergendo come un pilastro fondamentale dell&#39;attivit&agrave; biologica, come molte proteine sembrano partecipare, stabile o transitoriamente, nelle grandi assemblee di questa malattia. Analisi dell&#39;architettura di queste assemblee e loro interazioni collettore sono indispensabile per capire la loro funzione a livello molecolare. Genomica strutturale sforzi hanno promosso lo sviluppo di molte tecnologie per raggiungere la velocit&agrave; necessaria per studiare le singole proteine system-wide e interazione piccoli motivi ad alta risoluzione. Il cambiamento presente nella messa a fuoco verso grandi complessi multiproteici, in particolare negli eucarioti, ora chiede uno sforzo concertato similarmente per sviluppare e fornire nuove tecnologie che sono urgentemente necessarie per produrre in qualit&agrave; e quantit&agrave; la pletora di assembly multiproteici che formano il complexome e a studiare sistematicamente la loro struttura e funzione a livello molecolare. Gli attuali sforzi verso questo obiettivo sono riassunti e rivisto in questo contributo.

Ottenere una presa su complessi.

Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.

Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells.

Complexes multiprot&eacute;iques catalysent les fonctions biologiques vitales dans la cellule. Un objectif essentiel du projet SPINE2 est d&#39;aborder la biologie mol&eacute;culaire structurale de ces complexes multiprot&eacute;iques, en enr&ocirc;lant et le d&eacute;veloppement de technologies pour leur &eacute;tude habilitantes. Un pr&eacute;alable essentiel &eacute;mergente pour l&#39;&eacute;tude de sp&eacute;cimens biologiques complexes est leur surproduction recombinante. Nouveaux r&eacute;actifs et protocoles simplifi&eacute;es pour assembler rapidement des constructions de co-expression &agrave; cet effet ont &eacute;t&eacute; con&ccedil;us et valid&eacute;s. Le pipeline de haut d&eacute;bit mis en place &agrave; l&#39;IGBMC Strasbourg et la plate-forme d&#39;ACEMBL &agrave; l&#39;EMBL Grenoble utiliser les syst&egrave;mes de surexpression recombinant expression h&eacute;t&eacute;rologue de prot&eacute;ines et de leurs complexes. Extension de la technologie de plateforme ACEMBL pour inclure des h&ocirc;tes eucaryotes comme les insectes et les cellules de mammif&egrave;res ont &eacute;t&eacute; r&eacute;alis&eacute;e. Production efficace des complexes de grosse prot&eacute;ine &agrave; plusieurs composantes pour les &eacute;tudes structurales en utilisant le syst&egrave;me baculovirus/insecte cellule peut &ecirc;tre entrav&eacute;e par un d&eacute;s&eacute;quilibre stoechiom&eacute;trique des sous-unit&eacute;s produites. Une strat&eacute;gie de la polyprot&eacute;ine a &eacute;t&eacute; d&eacute;velopp&eacute;e pour surmonter ce goulot d&#39;&eacute;tranglement et a &eacute;t&eacute; mis en &oelig;uvre avec succ&egrave;s dans notre syst&egrave;me d&#39;expression baculovirus MultiBac pour produire des complexes multiprot&eacute;iques.

Robots, les pipelines, les polyprot&eacute;ines : activation multiprot&eacute;iques expression dans les cellules procaryotes et eucaryotes.

Complessi multiproteici catalizzano le funzioni biologiche vitali nella cella. Un obiettivo fondamentale del progetto SPINE2 &egrave; stato per affrontare la biologia molecolare strutturale di questi complessi multiproteici, l&#39;integrazione e lo sviluppo di tecnologie per il loro studio. Un prerequisito chiave emergente per lo studio di campioni biologici complessi &egrave; loro sovrapproduzione ricombinante. Romanzo reagenti e razionalizzati protocolli per assemblare rapidamente co-expression costrutti per questo scopo sono stati studiati e convalidati. La pipeline di alto-rendimento implementata IGBMC Strasburgo e della piattaforma ACEMBL presso l&#39;EMBL Grenoble utilizzano sistemi di sovraespressione ricombinante per l&#39;espressione eterologa delle proteine e dei loro complessi. Estensione della tecnologia piattaforma ACEMBL includere eucariotico host quali insetti e cellule di mammifero &egrave; stato raggiunto. Produzione efficiente di grandi proteine multicomponenti complessi per gli studi strutturali utilizzando il sistema baculovirus/insect cell pu&ograve; essere ostacolata da un squilibrio stechiometrico di subunit&agrave; prodotte. Una strategia polyprotein &egrave; stata sviluppata per superare questa impasse ed &egrave; stata implementata con successo nel nostro sistema di espressione di baculovirus MultiBac per la produzione di complessi multiproteici.

Robot, condutture, poliproteine: abilitazione multiproteici espressione in cellule di procariotiche ed eucarioti.

The initiation of gene transcription by RNA polymerase II is regulated by a plethora of proteins in human cells. The first general transcription factor to bind gene promoters is transcription factor IID (TFIID). TFIID triggers pre-initiation complex formation, functions as a coactivator by interacting with transcriptional activators and reads epigenetic marks. TFIID is a megadalton-sized multiprotein complex composed of TATA-box-binding protein (TBP) and 13 TBP-associated factors (TAFs). Despite its crucial role, the detailed architecture and assembly mechanism of TFIID remain elusive. Histone fold domains are prevalent in TAFs, and histone-like tetramer and octamer structures have been proposed in TFIID. A functional core-TFIID subcomplex was revealed in Drosophila nuclei, consisting of a subset of TAFs (TAF4, TAF5, TAF6, TAF9 and TAF12). These core subunits are thought to be present in two copies in holo-TFIID, in contrast to TBP and other TAFs that are present in a single copy, conveying a transition from symmetry to asymmetry in the TFIID assembly pathway. Here we present the structure of human core-TFIID determined by cryo-electron microscopy at 11.6â€‰Ã… resolution. Our structure reveals a two-fold symmetric, interlaced architecture, with pronounced protrusions, that accommodates all conserved structural features of the TAFs including the histone folds. We further demonstrate that binding of one TAF8-TAF10 complex breaks the original symmetry of core-TFIID. We propose that the resulting asymmetric structure serves as a functional scaffold to nucleate holo-TFIID assembly, by accreting one copy each of the remaining TAFs and TBP.

The architecture of human general transcription factor TFIID core complex.

L&#39;initiation de la transcription du g&egrave;ne de l&#39;ARN polym&eacute;rase II est r&eacute;gie par une multitude de prot&eacute;ines dans les cellules humaines. Le premier facteur de transcription g&eacute;n&eacute;rales pour lier les promoteurs de g&egrave;nes est le facteur de transcription IID (TFIID). TFIID d&eacute;clenche la formation de complexes paperasserie, fonctionne comme un co-activateur en interagissant avec les activateurs de la transcriptionnelles et lit les marques &eacute;pig&eacute;n&eacute;tiques. TFIID est qu&#39;un multiprotein m&eacute;gadaltons taille complexe compos&eacute; de TATA-bo&icirc;te-binding protein (TBP) et 13 facteurs associ&eacute;s &agrave; TBP (TAF). Malgr&eacute; son r&ocirc;le crucial, l&#39;architecture d&eacute;taill&eacute;e et le m&eacute;canisme de TFIID demeurent insaisissables. Histone pli domaines sont r&eacute;pandues dans les TAF, et structures t&eacute;tram&egrave;re et octam&egrave;re d&#39;histone semblables ont &eacute;t&eacute; propos&eacute;es dans TFIID. Un complexe fonctionnel noyau-TFIID a &eacute;t&eacute; r&eacute;v&eacute;l&eacute; dans les noyaux de drosophile, consistant en un sous-ensemble de TAF (TAF4, TAF5, TAF6, TAF9 et TAF12). Ces sous-unit&eacute;s de noyau sont cens&eacute;es &ecirc;tre pr&eacute;sents en deux exemplaires en holo-TFIID, contrairement &agrave; la PTB et autres TAF qui sont pr&eacute;sents en un seul exemplaire, en v&eacute;hiculant une transition entre la sym&eacute;trie et l&#39;asym&eacute;trie dans la voie d&#39;assemblage TFIID. Nous pr&eacute;sentons ici la structure de base-TFIID humaine d&eacute;termin&eacute;e par cryo microscopie &eacute;lectronique &agrave; 11.6 r&eacute;solution &Aring;. Notre structure r&eacute;v&egrave;le une double sym&eacute;trique, entrelac&eacute;e architecture, avec saillies prononc&eacute;es, qui tienne compte de toutes les caract&eacute;ristiques structurelles conserv&eacute;es des TAF, y compris les plis de l&#39;histone. Par ailleurs, nous d&eacute;montrent que la liaison d&#39;un complexe de TAF8-TAF10 rompt la sym&eacute;trie initiale de base-TFIID. Nous proposons que la structure asym&eacute;trique qui en r&eacute;sulte sert un &eacute;chafaudage fonctionnel &agrave; nucl&eacute;&eacute;e Assembl&eacute;e holo-TFIID, par accr&eacute;tion un copier chacun des TAF et PTB restants.

L&#39;architecture du complexe central pour transcription g&eacute;n&eacute;ral humain facteur TFIID.

L&#39;iniziazione della trascrizione del gene dalla RNA polimerasi II &egrave; regolata da una pletora di proteine in cellule umane. Il primo fattore di trascrizione generale per associare i promotori del gene &egrave; il fattore di trascrizione IID (TFIID). TFIID innesca la formazione di complessi pre-initiation, funziona come un coactivator interagendo con gli attivatori trascrizionali e legge marchi epigenetici. TFIID &egrave; che un complesso di multiprotein megadalton-dimensioni composto di TATA-box-binding protein (TBP) e 13 fattori associati TBP (TAF). Nonostante il suo ruolo cruciale, il meccanismo di montaggio di TFIID e dettagliata architettura resta sfuggente. Istone piega domini sono prevalenti in TAF e strutture tetramero e octamer istoni sono stati proposti nel TFIID. Un subcomplex funzionale core-TFIID &egrave; stato rivelato nei nuclei di drosofila, costituito da un sottoinsieme di TAFs (TAF4, TAF5, TAF6, TAF9 e TAF12). Queste subunit&agrave; nucleo si pensa siano presenti in due copie in holo-TFIID, contrariamente a TBP e altri TAF che sono presenti in una sola copia, trasportando una transizione dalla simmetria all&#39;asimmetria nella via dell&#39;Assemblea TFIID. Qui vi presentiamo la struttura del nucleo-TFIID umana determinata da crio-microscopia elettronica a 11,6 risoluzione &Aring;. La nostra struttura rivela una duplice simmetrica, interlacciata architettura, con marcate protrusioni, che ospita tutte le caratteristiche strutturali conservate di TAFs comprese le pieghe dell&#39;istone. Noi dimostrare ulteriormente che associazione di un complesso di TAF8-TAF10 rompe la simmetria originale del core-TFIID. Proponiamo che la struttura asimmetrica risultante funge da un&#39;impalcatura funzionale di nucleazione Assemblea holo-TFIID, di accrescimento di una copia ciascuno dei restanti TAFs e TBP.

Article	Year
New baculovirus expression tools for recombinant protein complex production. Journal of structural biology\| PubMed ID: 20178849	2010
Getting a grip on complexes. Current genomics\| PubMed ID: 20514218	2009
Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells. Journal of structural biology\| PubMed ID: 21419851	2011
The architecture of human general transcription factor TFIID core complex. Nature\| PubMed ID: 23292512	2013

Frederic Garzoni

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