the insect cell/baculovirus expression vector system for ...€¦ · the insect cell/baculovirus...
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The insect cell/baculovirus expression vector system for production of difficult to express
proteins and complexes
Integrated Structural Biology, IGBMC, Illkirch France
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©Kostyantyn SEMENCHENKO
Centre de Biologie Intégrative (CBI)
Instruct/FRISBI infrastructure, Strasbourg-node (FRANCE)
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Multi resolutionintegration
NMRBruno Kieffer
Protein ExpressionArnaud Poterszman, Bertrand Seraphin
Purification & biophysical characterizationCatherine Birck
Specialities available within the Instruct/FRISBI infrastructure, Strasbourg-node:
CrystallographyJean Cavarelli
Imaging CentreJean-Luc Vonesch
Cryo electron microscopy &tomographyBruno Klaholz, Patrick Schultz
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Transcription & DNA repair factors , Multi-subunit complexes.
TFIIH
Eukaryotic gene expression
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The transcription/DNA repair factor TFIIH
Nucleotide Excision Repair
Opens DNA around lesion (XPD)
Severe genetic disorders
Xeroderma pigmentosumCockayne’ syndromeTrichothyodystrophy
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Eukaryotic transcription factors, large multi-components complexes
Subunits often engaged in permanant protein-protein interactions:
In vitro reconstitution not always possible,
Co-expression which facilitates folding required.
Contain large and difficult-to-express proteins,
Sample preparation is often a challenge
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BEVSExpression of large intracelluar proteins (> 200kd)
Adapted for co-expression of multiple genes
Eukaryotic system:
Baculovirus Expression Vector System
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Principle
Infect invertebratesDouble-stranded DNA virus (135 kb)
The BV life cycle involves 2 distinct forms of virus
Occlusion Derived Virus (ODV) is present in a protein matrix(polyhedrin) and is responsible for the primary infection ofthe host while
Budded virus (BV) is released from the infected host cellslater during the secondary infection.
The polyhedrin matrix which protects viruses from theenvironment is not required for virus replication in a cellularsystem
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Principle
Cell lines grow well in suspension (27 °C, in Phosphate buffered medium, no CO2)Baculoviruses have a restricted host range and are safe to manipulateHigh levels of heterologous expression, production of toxic proteins
* non required for replication in a cellular system
Replace polyhedrin (PH) coding seq. with GOIUse strong late viral promoters (PH, P10) Protein expression 36-48 h post-infection
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An expression flowchart for BV expression
Bac
2Bac
HR Disabled viral DNA
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Baculovirus expression in practice
Clone the gene(s) of interest into a bacterial transfer vector
Amplify virus/Prepare a BIIC stock
Generate the recombinant virus Transfection/Co-transfection
Small scale expression assay
Optimization and large scale production
bacmid
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Homologous recombination in insect cells with disabled viral genome
Recombination inserts the foreign gene (GFP) into the viralDNA, restores the deleted gene, allowing virus replication.
No need for plaque selection (screening) for medium sizeinserts
Disabled viral DNA Co-transfection
An gene essential for virus replication has been deleted. BVparticles can only be formed if the truncation is bridged andrepaired by recombination with a suitable transfer vector.
Zhao, NAR 2003, Osz et al. 2014
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DsRed protein coding gene in the viral DNA.
Zhao, NAR 2003, Koleschnikoval et al. In prep
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A set of transfer vectors tailored for screening, multi-gene expression and standardization
Concatenation of expression units(restriction/ligation, SLIC…)
Vectors fusion(Cre/Lox in vitro recombination)
Introduction of a reporter gene (DsRed)
Inspired and compatible withmultibac approach (Berger, 2004)
By‐pass the need to preparebacmids
pAC8
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A set of transfer vectors adapted for screening and multi-gene expression
pAC8
pAC8 OpAC8 FpAC8 GpAC8 HpAC8 RpAC8 XpAC8 C
ProteinAFlagGST6xHisStrepTRXCBP
pAC8 F DsRedpAC8 G DsRedpAC8 H DsRedpAC8 R DsRed
FlagGST6xHisStrep
pAC8 0 GWpAC8 F GWpAC8 G GWpAC8 H GWpAC8 R GWpAC8 X GWpAC8 C GW
-FlagGST6xHisStrepTRXCBP
pAC8 0 GW loxPpAC8 F GW loxPpAC8 G GW loxPpAC8 H GW loxPpAC810H GW loxP
-FlagGST
6xHis10xHis
His
Strep
Flag
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Abdulrahman, Anal Bioch, 2009
The tag matters: the example of VDR
On column tag cleavageRXR capture assay
Production of a VDR (variant) to reconstitue a VDR/RXR heterodimer
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Strategies for production of multi-protein complexes
Separate expression of subunitspurify and mix; mix and purify
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In vitro reconstitution of RAR/RXR heterodimers
His-AB RXRF-RAR
Expression in Sf9 cells
Expression in E. coli
IMAC/GFPurification
with RXR ligand
Adsorb onto Flag affinity resin
Wash and incubatewith RAR ligand
incubate withexcess of purified
His-AB RXR
wash
elute with competitorpeptide
adsorb onto IMAC andelute to remove peptide
F-RAR
H6AB RXR
The partner required for solubility andstability can be a DNA or a ligand
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Strategies for production of multi-protein complexes
Separate expression of subunitspurify and mix; mix and purify
Contact area and polarity of various non obligate and obligate complexes. Thevertical ellipse denotes the aera-polarity space of weak transient interactions
No always possible. The interfaces in obligate complexes being generally large and hydrophobic.
Noreen & Thornthon, 2000
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Strategies for production of multi-protein complexes
Co-infection of insect cells by several viruses
Infection with a multigeneexpression virus
Separate expression of subunitspurify and mix; mix and purify
Co-expression
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Co-infection: a simple approach for co-expression
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Production of nuclear hormone receptor complexes
Rochel, et al. NSMB, 2011
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Reconstitution and functional analysis of the transcription DNA repair factor TFIIH
In vitro functional studies (founding paper (Tirode et al, 1999))
A PH domain in the p62 core-TFIIH subunit, dispensable for in-vitrotranscription but absolutly required for NER. Specifically interracts with theXPG endonucllease., (Gervais Nsmb 2004)
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p8/TTD-A is a subunit of TFIIH specifically involved in DNA repair specific and controls its intracellular concentration
Borrowed from Coin , Mol Cell et al 2006
XPB concentration probed with Ab-XPB
p34
p44
p62
C2
CN
N
p52 C
XPD
N
H1
M
C
XPB C
N
N
C
H1
H2
H2
C1
p8
TFIIH is a Tx/DNA repair factor
Transcription initiationNucleotide excision repair
Severe genetic disorders
Xeroderma pigmentosumCockayne’ syndromeTrichothyodystrophy
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Structure based analysis of p8/TTD-A complexes
Molecular basis of trichothiodystrophy
Kainov, et al. NSMB, 2008; Kainov et al., Acta D 2010
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Pair-wise and deletion analysis
Co-IPscore core-p52
p34F
p44p52
p62
XPB
1 2 3 4
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Systematic dissection of protein-proteininteractions: deletion analysis
Analysis of the protein interaction networkIdentification of key regulatory interactions
p34
p44
p62
C2
CN
N
p52 C
XPD
N
H1
M
C
XPB C
N
N
C
H1
H2
H2
C1 cycH
cdk7C
M
Np8
MAT1
Jawahri at al. 2002, Radu et al, in prep
core core-p52
p34F
p44p52
p62
XPB
1 2 3 4
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p89
p62
p44, p52
p34
Low yields, labour intensive, poor reproducibility
Co-infection with multiple viruses for reconstitution of complexes
# 50-100 g/L
Cell extract
IMAC
Affinity purification
Size exclusion
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Co-infection vs Multigene expression
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Co-infection
41
Controls Multigene expression
Idem for other MOIs
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Life sciences (Invitrogen)
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Multigene expression technologies: Combinatorial applications in protein protein complex production
Tools to streamline design of multigene expression recombinnant baculoviruses (Imre Berger)
Adapted from Abdulrhaman et al. Meth Mol Biol 2015)
Concatenation of several expression units
Fusion of single/multigene expression plasmids (LoxP)
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Concatenation of expression units(restriction/ligation, SLIC…)
Vectors fusion(Cre/Lox in vitro recombination)
Introduction of a reporter gene (DsRed)
Inspired and compatible withmultibac approach (Berger, 2004)
By‐pass the need to preparebacmids
pAC8
A set of transfer vectors tailored for screening, multi-geneexpression and standardization
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Insertion of an expression cassette into the multiplication module
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pSPL Multibac, (Fitzgerald, Nat Methods 2006)
Cre-LoxP recombination in vitro
(A)
(D)
Cre recombinase binds to the loxP sites on both the donor vector and the acceptor vector, cleaves the DNA, and covalently attaches itself to the DNA which leads to strand exchange and concatenation.
•34-bp recognition sequence•13-bp inverted repeats flanked by 8-bp spacer region
8-bp overlap region
loxP sequence
Inverted region Inverted region
loxP
loxP
loxP
pAC8 -GFP
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Co-infection vs Multigene expression: a first exemple
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Expression of ternary and quaternary complexes witha single virus: a problem of DNA synthesis
Flag-affinity
Strep-affinity
Elution withCompetitor-peptide
Elution with Desthiobitin
pBac8 Multi-pSPL Fusion
Ternary complex Quaternary complex
(Fitzgerald, Nat Methods 2006)
D
ABC
ABC
A: strep-tagD: Flag-tag
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Production of core-TFIIH with a single virus
6 subunits: XPB, p62, p52, p44, p34, p8/TTDA (+ DsRed)
Yield : 0.5 mg/L
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Reconstitution and in vitro TFIIH assays revisited
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XPD, a DNA helicase exclusively devoted to repair
(Abdulrahman et al, PNAS 2013, Kupper et al., Plos Biology 2014)
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ConclusionsIllustration of strategies used for several projects at IGBMC for production of human multi-protein complexes.
Separate expression of subunits and in vitro reconstitution with purified proteins or from crude extracts is an option.
In most cases, isolated proteins are difficult-to express/ manipulate and co-expression is required to for proper folding
Co-infection of insect cells by several viruses has severaladvantages (screening, dissection of PPI networks)
Infection with a multigene expression virus often leadsto better yields/improved consistency and mandatoryfor large complexes (3 and more subunits)
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Support
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Laura RaduOlga KoleschnikovaWassim AbdulramanDennis KainovAnne Maglott-RothMarc Vitorino
Isabelle BillasJean CavarelliNatacha Rochel
Dino Moras
Jean Marc Egly & F Coin s groupE. CompeC. Braun
Patrick Schultz’s groupG Papai
Alain Van Dorsselaer/Sahara Sanglier(LSMBO, Strasbourg)
J. Marcoux
Jochen Kupfer/Karoline Kisker(University Wurtzbourg)
IGBMC common facilities: Isabelle Kolb-Cheynel and Nathalie Troeffer, Jean-Marie Garnier
Structural Biology Platform
Acknowledgements