The insect cell/baculovirus expression vector system for production of difficult to express

proteins and complexes

Arnaud.Poterszman@igbmc.fr

Integrated Structural Biology, IGBMC, Illkirch France

©Kostyantyn SEMENCHENKO

Centre de Biologie Intégrative (CBI)

Instruct/FRISBI infrastructure, Strasbourg-node (FRANCE)

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

Transcription & DNA repair factors , Multi-subunit complexes.

TFIIH

Eukaryotic gene expression

The transcription/DNA repair factor TFIIH

Nucleotide Excision Repair

Opens DNA around lesion (XPD)

Severe genetic disorders

Xeroderma pigmentosumCockayne’ syndromeTrichothyodystrophy

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

BEVSExpression of large intracelluar proteins (> 200kd)

Adapted for co-expression of multiple genes

Eukaryotic system:

Baculovirus Expression Vector System

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

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

An expression flowchart for BV expression

Bac

2Bac

HR Disabled viral DNA

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

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

DsRed protein coding gene in the viral DNA.

Zhao, NAR 2003, Koleschnikoval et al. In prep

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

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

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

Strategies for production of multi-protein complexes

Separate expression of subunitspurify and mix; mix and purify

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

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

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

Co-infection: a simple approach for co-expression

Production of nuclear hormone receptor complexes

Rochel, et al. NSMB, 2011

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)

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

Structure based analysis of p8/TTD-A complexes

Molecular basis of trichothiodystrophy

Kainov, et al. NSMB, 2008; Kainov et al., Acta D 2010

Pair-wise and deletion analysis

Co-IPscore core-p52

p34F

p44p52

p62

XPB

1 2 3 4

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

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

Co-infection vs Multigene expression

Co-infection

41

Controls Multigene expression

Idem for other MOIs

Life sciences (Invitrogen)

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)

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

Insertion of an expression cassette into the multiplication module

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

Co-infection vs Multigene expression: a first exemple

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

Production of core-TFIIH with a single virus

6 subunits: XPB, p62, p52, p44, p34, p8/TTDA (+ DsRed)

Yield : 0.5 mg/L

Reconstitution and in vitro TFIIH assays revisited

XPD, a DNA helicase exclusively devoted to repair

(Abdulrahman et al, PNAS 2013, Kupper et al., Plos Biology 2014)

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)

Support

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