Prsentation PowerPoint

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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

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Identification, production and purification
Identification methods Purification
strategies Single gene expression and in vitro
assembly Co-expression techniques
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Recombinant protein production
Prokaryotic E. coli, Anabaena .... Eukary
otic Yeast Insect cells Mammalian Cell
free systems E coli Wheat germ, Insect
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Recombinant protein production in E. coli
Strain XL1-Blue, BL21, . Expression
vector antibiotic resistance replication
origin promoter
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Lac Promoter

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pBAD vectors

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pET vectors
BL21 DE3
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pET vectors
T7 promoter
ColE1
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Native and tagged proteins
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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
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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
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Why HEK.EBNA Cells? Advantages

• In comparison to other eukaryotic expression
  systemsthe HEK.EBNA Expression System is
  rapidfrom 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

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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)
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Transient transfection
Vector purification 20 l fermentation 30 g cell
paste 15-30 mg plasmid
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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)
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The BaculoVirus Expression System (BEVS)
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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
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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)
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Baculovirus Life Cycle
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

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Baculovirus Polyhedrin Promoter

• biotechnologists have utilized this system
• polyhedrin promoter is strong
• dont 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

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Baculovirus System

• 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

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Post-Translational Modifications

• 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)...
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Disadvantages of Baculovirus Expression System

• 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

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Safety considerations

• 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

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Flowchart for Baculovirus Expression
Clone the gene(s) of interest into a bacterial
transfer vector
Generate the recombinant virus Transfection/Co-tra
nsfection Small scale expression assay
Prepare a high titer virus stock lt108 pfu/ml
Optimization of the expression conditions and
large scale production
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Basic concepts

• 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 (MOIlt0.1), hight for protein
  production (1ltMOI)

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What is needed to express a protein ?

• 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

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Homologous recombination
The transfer vector and the viral DNA are
co-transfected
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A transfer vector
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Transposition in E. Coli
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What is needed ?

• 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

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What is a bacmid ?
AcMNPV genome
E.coli plasmid
Bacmid
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Recombinant Bacmid
Bacmid
lacZ will be disrupted - white colonies on X-Gal
Helper Plasmid with transposition genes
Donor Plasmid
Recombinant Bacmid
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A donnor plasmid
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Single gene baculovirus transfer vectors
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
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Flowchart for Baculovirus Expression
Clone the gene(s) of interest into a bacterial
transfer vector
Generate the recombinant virus Transfection/Co-tra
nsfection Small scale expression assay
Prepare a high titer virus stock lt108 pfu/ml
Optimization of the expression conditions and
large scale production
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Homologous recombination in insect
cells (FlashBac, Pharmingen, tri-ex)
Homologous Recombinaison in E.Coli (Bacmids)
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Day -5
Bacmids
Cloned cDNA
transformation in E. coli DH10bac/Multibac select
ion 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
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Upscaling and purification issues
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
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Titering a virus stock
TidiousInfected cells do not divide - Plaque
assay - end point dilution - rt pcr -
viability assays
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Optimization
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, 10 Very
important for co-infections experiments - cell
line/media of choice Sf9, Sf21, H5 with or
without serum
Small scale experiments 4 ml cultures
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Towards simplification and automation
Culture Lysis Centrifugation
24-Deep well block (3ml culture)
Purification
Plaque 96-wells (batch purification)
Coomassie Analysis
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Nucleic acid synthesis
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)
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Production par transcription in vitro / PolT7
linéarisation
pUCT7
polT7 NTPs
Matrices plasmidiques
RNA transcrit
linéarisation
pRZ/pHDV
polT7 NTPs Mg
RNA dintérêt (3 franc) ribozyme
Ou oligos
polT7 NTPs
RNA court transcrit
Anne Catherine Dock-Bregreon