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Strategies. Non-obligatory/Obligatory Transient/stable. Yes. No. Produce (and purify) the proteins independently and reconstitute the complex in vitro. Co-expression is mandatory. Co-expression in E. coli or in insect cells Purification of Endogenous complexes. Single gene expression

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Strategies

Non-obligatory/Obligatory

Transient/stable

Yes

No

Produce (and purify) the

proteins independently

and reconstitute the complex in vitro

Co-expression is mandatory

Co-expression

in E. coli or in insect cells

Purification of Endogenous complexes

  • Single gene expression

    • E. coli

    • Insect cells baculovirus

    • Yeast

    • Mammalian


Identification, production and purification

Identification methods

Purification strategies

Single gene expression and in vitro assembly

Co-expression techniques


Recombinant protein production

Prokaryotic

E. coli,

Anabaena

....

Eukaryotic

Yeast

Insect cells

Mammalian

Cell free systems:

E coli

Wheat germ,

Insect


Recombinant protein production in E. coli

Strain: XL1-Blue, BL21, ….

Expression vector

antibiotic resistance

replication origin

promoter




Pet vectors
pET vectors

BL21 [DE3]


Pet vectors1
pET vectors

T7 promoter

ColE1



Yeast expression

PCR, cloning, transformation

S. cerevisiae P. pastoris

Copper inducible Methanol inducible

Replicative Integrative

Intracellular

Secretion vectors

Different tag systems

Streamlining of workflow

by recombinational in vivo cloning in S. cerevisiae


EBNA-1 protein drives episomal replication ofori-P containing plasmids

integrated Ad5

E1a/E1b fragment

in HEK 293 cells

enhances trans-

cription of CMV

promotor driven

transgene

Why HEK.EBNA Cells? The Principle

EBNA-1/ori-P based expression in Human Embryonic Kidney (293) cells (293 stably transformed with EBNA-1 gene)

The cell line is available from ATCC and, until recently, also from Invitrogen


Why HEK.EBNA Cells? Advantages

  • In comparison to other eukaryotic expression systemsthe HEK.EBNA Expression System is rapid:from gene to protein in 4-6 weeks

  • The cells can be grown adherently and in serum-free suspension culture

  • It can be applied to generate stable cell lines (pools/ clones) and in transient mode on small and large scale

  • In transient mode not only secreted and membrane-bound, but also intracellular proteins can successfullybe expressed


HEK.EBNA Expression Vectors

  • Basic vector (alsoGateway™ adapted)

  • Can be decorated withN- or C-terminal tags, heterologous leadersequences

  • Co-expression of e.g. GFP via IRES element

  • Selectable marker for generation of stable cell line

Commercially available HEK.EBNA vectors: pREP4 and pCEP4 (Invitrogen)


25

10

Cell density in 3.6 volume

prior to transfection

9

Cell density after addition

of 1.4 l transfection mix

20

8

cells/ml]

Cell density after addition

7

of 5 l growth medium

E. Coli DH5

5

15

6

product titer [mg/l]

5

cell density [ x 10

10

4

3

5

2

1

0

0

0

20

40

60

80

100

120

140

160

180

time [h]

cell density

product titer

Transient transfection

Vector purification

20 l fermentation

30 g cell paste

15-30 mg plasmid


For Secreted Proteins: affinity chromatography on antibody or Protein A column -tag dependent-

For Intracellularly Expressed Proteins: his and/or his-Strep tag  Ni-chelate and/or Streptactin column

(oxford)



What are baculoviruses ?

Baculoviruses are a group of viruses found mostly on insects

They are rod-shaped (latin baculum = stick), 40-50 nm in diameter and 200-400 nm in lengh

Double stranded , covalently closed and circular DNA (80 – 200 kbp)

Trichoplusia ni

Spodoptera frugiperda


In cell culture or when multiplying within an insect host, baculoviruses

Form so called virions, also referred to as non-occuded or budded virus (BV)

For long-term survival occlusion bodies (OB) or polyhedra are formed.

Para-crystalin matrix, composed of polyhedrin (50% of the total

protein mass)


Baculovirus life cycle
Baculovirus Life Cycle baculoviruses

Early phase (0-6 post infection)

  • Enters the cells by endocytosis and the nucleocapsid rapidly migrate to nucleus

  • Viral DNA is realeased inside the nucleus and early gene expression starts

Late phase (6-20 post infection)

  • Extensive DNA replication and subsequent production of budded virus particles after 12 p.i.

  • Projeny nucleocapsids leave nucleaus and axquire an envelope as the leav ethe cytoplasm as budded virus

• 36-48h post infection

• polyhedrin protein traps

many virions into polyhedron

“package”

• polyhedron is stable

Very Late phase (after 20 hrs post infection)

  • Decrease in the formation of budded virus (BV)

  • Nucleocapsids acquire an envelope inside the nucleus to form multiple nuclear polyhedrosis virus (MNPVs)

  • MNPVs are embodied in the polyhedrin matrix and fromation of occlusion bodies

  • Accumulation of the p10 protein in fibrous material

  • Cell lysis begins 60_72 hours post infection


Baculovirus polyhedrin promoter
Baculovirus Polyhedrin Promoter baculoviruses

  • biotechnologists have utilized this system

    • polyhedrin promoter is strong

    • don’t need polyhedron “package” in lab

    • replace polyhedrin coding seq. with GOI

    • Express lots of protein 36-48 h post-infection

  • Protein production in a eukaryotic host

    • Proper folding

    • Post-translational modifications


Baculovirus system
Baculovirus System baculoviruses

  • Virus utilized

    • AcMNPV

      • Autographa californica multiple nuclear polyhedrosis virus

    • A. californica = alfalfa looper

    • AcMNPV infects 30+ insects

    • Commonly used cell line

      • Fall armyworm - Spodoptera frugiperda (Sf9)

      • Polyhedrin promoter very active


Post translational modifications
Post-Translational Modifications baculoviruses

  • Possible modifications

    Insect cells expresssing heterologous proteins in the BVES appear to be able to cope with post-translational modifications: Signal peptide cleavage, Phosphorylation, Glycosylation, Acetylation, sulfation, disulfide bond formation...

  • Limitations

    Kind or degree of modification may not always be identical to the ones found in the original species or tissue backgound of the gene

For structural biology post translational modification and especially glycosylation are problematic (source of heterogeneity)...


Disadvantages of baculovirus expression system
Disadvantages of Baculovirus Expression System baculoviruses

  • Insects cells have a large doubling time

  • Baculovirus system kills the cells - not continuous production

  • Some proteins not modified correctly

  • Insect cell culture is expensive and time consuming


Safety considerations
Safety considerations baculoviruses

  • Personal risk or contamination of mammalian cells

    - baculovirus can enter mammalian cells but not reach

    nuclei or start gene expresion

    - baculoviruses are considered to pose no additional safety risk

    and are classified as Class I (like E. Coli K12)

  • Contaminating other insect cells

    - although their host range is fairly limited, baculoviruses are able to enter other other insect cells but not to replicate their DNA or enter late infection stages

    - care must be taken not to contaminate other insect work (baculoviruses can be used as pesticides)

    Like other recombinant organisms, baculoviruses are potential biohazards and care must be taken regarding their distribution and containment


Flowchart for baculovirus expression
Flowchart for Baculovirus Expression baculoviruses

Clone the gene(s) of interest into a bacterial transfer vector

Generate the recombinant virus Transfection/Co-transfection

Small scale expression assay

Prepare a high titer virus stock <108 pfu/ml

Optimization of the expression conditions and large scale production


Basic concepts
Basic concepts baculoviruses

  • Several cell lines (Sf9, Sf21, H5) and media (TNMFH, SF900-II, Express five...) are availible

  • Cells can be grown on monolayers (T25, T175) or in suspension (Deep Well, Spinner Flask, Erlenmeyer, Wave bag, Bioreactor)

  • Pfu: plaque forming unit

    Pfu/ml is the measure of viral titre, equivalent to the concentration of viruses

  • MOI: multiplicity of infection

    Number of viruses per cell. Usually low for virus amplification (MOI<0.1), hight for protein production (1<MOI)


What is needed to express a protein
What is needed to express a protein ? baculoviruses

The expression unit

  • Strong promoter: p10 or PH

  • Kozak sequence:

  • Gene of interest

  • Terminator

On both sides, elements that will allow the integration of the expression unit(s) into the viral genome

  • Segment from viral genome for homologous recombination

    in insect cells between the transfert vector and the viral DNA

  • Transposons (Tn7L and Tn7R) recombination sites (LoxP)

    when a bacmid is to be used


Homologous recombination
Homologous recombination baculoviruses

The transfer vector and the viral DNA are co-transfected


A transfer vector
A transfer vector baculoviruses



What is needed
What is needed ? baculoviruses

  • The target gene cloned into the transfer plasmid

    • donor plasmid

  • Bacmid formed by integration of E. coli plasmid into AcMNPV genome

    • double crossover event

    • ori of replication, select. marker, etc.

  • 3rd plasmid provides proteins to move gene of interest into bacmid

    • Helper plasmid


What is a bacmid
What is a bacmid ? baculoviruses

AcMNPV genome

E.coli plasmid

Bacmid


Recombinant bacmid
Recombinant Bacmid baculoviruses

Bacmid

lacZ will be disrupted -

white colonies on X-Gal

Helper Plasmid

with transposition genes

Donor Plasmid

Recombinant Bacmid


A donnor plasmid
A donnor plasmid baculoviruses


Single gene baculovirus transfer vectors baculoviruses

Numerous transfer vectors adapted are availible

- Recombination in E. coli or in insect cells

- Ligation based or Gateway based cloning

- Expression of Native, N- or C- terminal tagged proteins

GST

Native

Flag

Hemagglutinin

His6

His6

CBP

Gateway

Kozak

ATG

pBacGW

F. Klein, S. Schochat, Y. Trottier, A. Poterszman, L. Salim, D. Busso

Multi-system: TriEx or Gateway


Flowchart for baculovirus expression1
Flowchart for Baculovirus Expression baculoviruses

Clone the gene(s) of interest into a bacterial transfer vector

Generate the recombinant virus Transfection/Co-transfection

Small scale expression assay

Prepare a high titer virus stock <108 pfu/ml

Optimization of the expression conditions and large scale production


Homologous recombination in insect cells baculoviruses

(FlashBac, Pharmingen, tri-ex)

Homologous Recombinaison in E.Coli

(Bacmids)


Day -5 baculoviruses

Bacmids

Cloned cDNA

transformation in E. coli DH10bac/Multibac

selection of recombinant bacmids

(white colonies)

bacmid preparation (Mini-prep)

Transfer vectors

co-transfection of the transfer vector with the viral genome in insect cells

transfection of the bacmid

in insect cells

Day 0

Day 0

Day 7-10

Initial virus stock

P0, # 4 ml

7 Days/round

Amplification, P1, P2

purification, titration

Small scale analysis

(6 Well plate)

Day 12-15

Optimization

Large scale production


Upscaling and purification issues baculoviruses

High titer virus stock

Sf9, Hi5, Tni cells in exp phase

T = 0 h Infection

T= 48 or 72 Harvest

Optimization of the expression conditions and large scale production


Titering a virus stock baculoviruses

TidiousInfected cells do not divide - Plaque assay - end point dilution - rt pcr - viability assays


Optimization baculoviruses

For infections cells in exponential growth phase are required. infect cells a 0.5 to 2.0 1O6 cell/ml T =27 °C Monolayers or suspension (Deep Well, Spinner, Bottles..)

Optimization fo the culture conditions - harvest time post-infection: 48, 72, 96 hrs - multiplicities of infection: 0.1, 1, 5, 10Very important for co-infections experiments - cell line/media of choice: Sf9, Sf21, H5 with or without serum

Small scale experiments: 4 ml cultures


Towards simplification and automation baculoviruses

Culture

Lysis

Centrifugation

24-Deep well block

(3ml culture)

Purification

Plaque 96-wells

(batch purification)

Coomassie Analysis


Nucleic acid synthesis baculoviruses

Chemical synthesis:

DNA or RNA oligonucleotides (up to 40 bps)

Introduction of modified bases

In vitro synthesis:

DNA :DNA polymerase by PCR (100-500 bp for em)

RNA: T7 RNA Pol

In vivo synthesis:

tRNAs, DNA (100-500 bp for em)


Production : par transcription baculovirusesin vitro / PolT7

linéarisation

pUCT7

polT7

NTPs

Matrices

plasmidiques

RNA

transcrit

linéarisation

pRZ/pHDV

polT7

NTPs

+ Mg

RNA d’intérêt (3’ franc) + ribozyme

Ou oligos

polT7

NTPs

RNA court

transcrit

Anne Catherine Dock-Bregreon


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