Purificazione di proteine umane da
animali
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Basse rese
Difficili da purificare
Costoso
Possibilita’ di malattie
How can we synthesise
human proteins?
• Use bacterial cells
• Human gene lacks
• Bacterial promoter
• Bacterial terminator
• Bacterial ribosome binding site
• Cannot deal with introns
Dealing with introns
DNA
RNA
Protein
RNA
Reverse
transcriptase
DNA
Protein Expression in E. coli
Advantages
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Inexpensive
Easy to manipulate
Well characterized
Grows quickly
rProtein up to 50%
total protein
and
Disadvantages
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Post-transcriptional modification
Post-translational modification
Poor folding
Proteolysis
N-terminal Methionine
Complicated purification
Lack of efficient secretion
Possible toxicity
E. coli Expression Vector
Promoter
Selectable
Marker
E. coli Promoters
Weickert, et al., 1996
E. coli Expression Vector
Promoter
SD AUG
Stop
Transcriptional
Terminator
Repressor
Selectable
Marker
Ori
What if expression is low?
Optimizing Expression
• Examine codon usage
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–
–
–
Decrease message stability
Premature termination of transcription
Premature termination of translation
Frameshifts, deletions, and misincorporation
Codon Frequency in E. coli
What if expression is low?
Optimizing Expression
• Examine codon usage
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Minimize GC at 5’
Add terminator
Add fusion and/or tags
Growth conditions
Combined approach
Expression of Fusion Proteins
• Increase expression
• Ease of purification
• Ease of detection
• Increase solubility
• Increase stability
Examples of Fusions/Tags
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Hexahistidine-tag
GST
MBP
CBP/Intein
Arg-tag
S-tag
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Ni affinity
GSH
Amylose
Chitin
Ion-Exchange
RNAse
Insoluble Proteins
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Growth Temp
Media
Expression rate
Chaperones
Coexpression of subunits
Express as polymer
Redox potential
Periplasmic expression
Fusion
Tags
Express as a fragment
Denature and renature
Combined approach
Improving Protein Stability
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Protease inhibitors
Protease-minus host
Periplasmic expression
Growth temperature
Combined approach
MANIPOLAZIONE DELL’ESPRESSIONE GENICA
NEI PROCARIOTI
-PROTEINE DI INTERESSE TERAPEUTICO E COMMERCIALE
POSSONO ESSERE PRODOTTE IN E. coli CON TECNICHE
DNA RICOMBINANTE
-PROMOTORE
-SEQUENZE LEGANTI I RIBOSOMI ( 6-8 nt Seq. di Shine Dalgarno)
-NUMERO COPIE DEL GENE CLONATO
-LOCALIZZAZIONE FINALE PROTEINA
-STABILITA’ PROTEINA IN CELLULA OSPITE
GENI IN PROCARIOTI POSSONO AVERE
-ESPRESSIONE COSTITUTIVA
-ESPRESSIONE REGOLATA (es. lac operon)
NELLA PRODUZIONE DI PROTEINE ETEROLOGHE IN
BATTERI VENGONO UTILIZZATI SPESSO PROMOTORI
FORTI E REGOLABILI
UNA PRODUZIONE CONTINUA PROVOCA:
-INIBIZIONE FUNZIONI CELLULA
-PERDITA ENERGIA
-PERDITA PLASMIDE
Bottlenecks to efficient protein expression in E. coli
l Inefficient transcription
No or little protein synthesized
u Promoter choice and design
l Inefficient translation
No or little protein synthesized
u Codon usage
u Transcript stability
u Transcript secondary structure
l Inefficient folding (cytoplasmic or periplasmic)
Aggregation or degradation
u Improper secondary, tertiary or quaternary structure formation
u Inefficient or improper disulfide bridge formation
u Inefficient isomerization of peptidyl-prolyl bonds
l Inefficient membrane insertion/translocation
l Toxicity
Cell death
Aggregation or degradation
Folding chaperones in de novo folding
J
K
K
TF
J
ATP
3'
5'
GrpE
ATP
GrpE
ADP
ADP
Native
Aggregate
ADP
ATP
GroEL
GroES
GroEL-GroES co-expression and low temperatures
improve leptin folding
However, this strategy does not always work
PROTEINE DI FUSIONE
-PER EVITARE DEGRADAZIONE DI PICCOLE PROTEINE
ETEROLOGHE QUESTE VENGONO PRODOTTE COME
PROTEINE DI FUSIONE CON UNA PROTEINA STABILE
DELL’ORGANISMO OSPITE.
-I DUE cDNA DEVONO ESSERE FUSI MANTENENDO LA
CORRETTA CORNICE DI LETTURA
cDNA di interesse
MCS
MBP o
GST
PROMOTORE
REGOLABILE
cDNA di interesse
GST o MBP
UTILIZZATE PER PURIFICAZIONE
MCS
MBP o
GST
SITO DI TAGLIO
PER PROTEASI
PROTEINA DI
INTERESSE
INDUZIONE DI
ESPRESSIONE
PROTEINA DI
FUSIONE
(PROMOTORI
REGOLABILI)
TRASFORMAZIONE IN
BATTERI
Promotore
“lac”
gene
MalE
Proteina di fusione
MBP
pMAL
cDNA
di interesse
Resina
con legato
maltosio
Eluizione
Proteina di fusione purificata
pGEX
GST comes from
Schistosoma mansoni
•IPTG
induction
tac
•High level
expression
GST
Foreign gene
PURIFICATION OF GST
FUSION PROTEINS
PURIFICATION
• EASY
• AFFINITY CHROMATOGRAPHY
PURIFICATION
DETAILS
• GROW SAY 1L CULTURE TO MID LOG
PHASE
• ie OD260 = 0.4 – 0.7
• SPIN DOWN CELLS
• SONICATE IN PRESENCE OF
PROTEASE INHIBITORS
• POUR LYSATE OVER GLUTAHIONE
SEPHAROSE BEADS IN A COLUMN
GLUTATHIONE SEPHAROSE
glutathione
SEPHAROSE
FUSION PROTEIN
FOREIGN PEPTIDE
GST
FUSION PROTEIN BOUND TO
GLUTATHIONE SEPHAROSE
FOREIGN PEPTIDE
GST
glutathione
SEPHAROSE
PURIFICATION
• WASH COLUMN EXTENSIVELY
• ELUTE WITH REDUCED
GLUTATHIONE
• RESULTS IN PURE GST FUSION
PROTEIN
COMPETITIVE ELUTION WITH
GLUTATHIONE
SEPHAROSE
RESULT OF AFFINTY PURIFICATION AND
REMOVAL OF GST MOIETY
pure fusion protein
+ glutathione
foreign peptide
+
GST
protease
dialyse
pure fusion
second
glutathione
column
pure foreign
peptide in flow
through GST sticks
pQE VECTORS (Qia Express)
• Hex-histidine tag system
• Produce peptides with 6 histidines fused to
N or C terminus
• Allows Nickel Chelate Affinity
Chromatography
pQE VECTORS (Qia Express)
• Promoter
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–
–
–
engineered from phage T5 + lac operator
2 operator sites
IPTG inducible
Expression in host containing multiple copies
of pREP4 which has lacI
pQE VECTORS (Qia Express)
• Interaction between Ni2+ resin called NTA
is very strong and chemically resilient
– every Ni2+ binds 2 his residues in a nonconformation dependent manner
– therefore resists strong denaturants eg 6M
guanidium HCl
pQE VECTORS (Qia Express)
• Elution
– competitive with imidazole
O
N
N
N
Histidine
N
N
Imidazole
pQE VECTORS (Qia Express)
• Removal of His tag?
– not necessary usually
– many hundreds of proteins purified with no
effect on structure
– not immunogenic
PROTEINE DI INTERESSE TERAPEUTICO IN PROCARIOTI:
-RISCHIO CONTAMINAZIONE VIRALE NULLO
-RISCHIO ALLERGIE NULLO (vengono prodotte proteine umane)
PRODUZIONE DI INSULINA UMANA IN E. coli
-70 MAIALI PER 1 PAZIENTE PER UN ANNO
-E. Coli NON SA MODIFICARE premRNA EUCARIOTICI
E PRODURRE MODIFICHE POST-TRASCRIZIONALI
SINTESI INSULINA IN CELLULA PANCREATICA
CATENA A
CATENA B
30 aa
21 aa
Unite da ponti S-S
ESONE 2
ESONE 1
PREPROINSULINA
PEPTIDE SEGNALE
PROINSULINA FORMA S-S
INSULINA
IN APPARATO DEL GOLGI
UN ENZIMA RIMUOVE 33aa
PRODUZIONE DI INSULINA
RICOMBINANTE IN BATTERI
-Plasimidi separati codificano per
Catena A e B
-promotore trp e alcuni codoni iniziali trp
-seq per il trp sono eliminate con
trattamento con bromuro di cianato
-catene mescolate assieme e tramite un
processo chimico si formano legami S-S
PRODUZIONE ORMONE DELLA CRESCITA UMANO
IN E. Coli
-Peptide di 191 aa
-Carenza provoca nanismo
-GH da animali non è efficace sull’uomo
-80 ipofisi di cadaveri umani per un paziente per un anno (alto
rischio infezioni)
PRODUZIONE DI GH
RICOMIBINATE IN BATTERI
SALMONELLA
• Expression host
• Live vaccine delivery
SALMONELLA
• Salmonella is itself a pathogen – S.typhi causes typhoid
• It is possible to vaccinate aganst with attenuated strains
• Attenuated Salmonella can persist in the gut and
disseminate
• Induces mucosal & systemic cellular & humoral responses
• It has potential to be engineered as one shot, multivalent
vaccines
SALMONELLA
• Recognises E.coli promoters and origins of replication
– therefore existing vectors can function
• Several ways of attenuating Salmonella have been
discovered
EXPRESSION SYSTEMS
MOST USE PLASMIDS
– PROBLEMS
• INSTABILITY
• TOXICITY
• pIP-pET DUAL PLASMID
• NirB-ANAEROBIC INDUCIBLE
• BALANCED LETHAL
pIP-pET DUAL PLASMID
foreign
antigen
pL
T7
promoter
c1ts
pIP
T7 RNA
polymerase
pET
AmpR
kanR
c1ts= l repressor active 28°C, inactive at 37°C
pL = l left promoter
pTECH VECTORS
• THESE USE THE NIRB PROMOTER
• NIRB ENCODES NADH-DEPENDENT
NITRITE REDUCTASE
• NIRB INDUCED IN ANAEROBIC
CONDITIONS eg GUT & TISSUES
pTECH VECTORS
Khan made this vector
GST
NirB
promoter
tetanus
toxoid
pTECH
AmpR
Oral immunisation,
single dose in mice
-protected against
Salmonella
Tetanus toxin
BALANCED – LETHAL
SYSTEM
• OTHER SYSTEMS DESCRIBED CARRY ANTIBIOTIC
RESISTANCE-UNDESIREABLE
• THESE VECTORS COMPLEMENT LETHAL
DELETION IN HOST
• GENE FOR B-ASPARTATE SEMI-ALDEHYDE
DEHYDROGENASE OR asd
• asd MUTANTS HAVE ABSOLUTE REQUIREMENT
FOR DIAMINOPIMELIC ACID (DAP) A
CONSTITUENT OF THE CELL WALL
• THERE IS NO DAP IN MAMMALS
Balanced Lethal
foreign gene
trc
promoter
pYA292
asd
asd complements asd D host & is thus stable
Heterologous Expression in Yeast
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Codon usage is closer to human
Glycosylation of exported proteins
Purification of proteins from the medium
Ease of transformation
Ease of growth
EXPRESSION IN
PICHIA PASTORIS
PICHIA PASTORIS
• USES ALCOHOL OXIDASE 1 (AOX1)
PROMOTER
• AOX1 IS INDUCIBLE BY METHANOL
AND GENE IS EXPRESSED AT VERY
HIGH LEVELS
• THERE ARE THREE BASIC STEPS
STEP1
• CLONE GENE OF INTEREST INTO
SHUTTLE VECTOR DOWNSTREAM
OF AOX1 PROMOTER IN E. coli
TT
gene of
interest
HIS4+
AOX1
promoter
3’ AOX1
STEP2
• TRANSFORM HIS- PICHIA PASTORIS YEAST
WITH PLASMID. SELECT FOR HIS+ STABLE
INTEGRANTS DISRUPTED IN THE AOX1 LOCUS
STEP2
TT
gene of
interest
HIS4+
AOX1
promoter
3’ AOX1
P.pastoris chromosome
INTEGRATION
3’ AOX1
pAOX1
gene of
interest
TT 3’ AOX1
• Pichia pastoris production of single-chain
antibody fragments (scFv)
• A CASE STUDY
1. PLACE scFv cDNA in vector pPIC9K
pPIC9K
PLACE scFv cDNA in vector pPIC9K
pAOX1
scFv cDNA
a-mating
type
secretion
signal
ALL RECOMBINANT STEPS DONE IN E.coli
His 6 tag
scFv expression in P. pastoris
2. Transform HIS- P. pastoris by
electroporation
Select on minimal media
3. Check medium for product after methanol
induction.
POSITIVE
scFv expression in P. pastoris
4. Large scale up
• 5 litres capacity stirred reactor
• 4L medium plus 400 ml starter culture
• Grow 17h @ 30oC in glycerol
• Dense
• Keep pH stable @ 6.0
• Induce 48 h with methanol
• Harvest culture medium
• Adjust pH to 7.4 and Affinity Purify by Nickel
Chelate Chromatography
YIELDS
• For scFV antibody 250 mg per L
OTHER EXAMPLES
• highest yield
– tetanus toxin frag C
(INTRACELLULAR)
– a amylase
(SECRETED)
12g per L
2.5g per L
CAN WORK ON INDUSTRIAL SCALE
YIELDS
PRODUCT
YIELD g per L
ENZYMES
Invertase
2.3
a amylase
2.5
ANTIGENS
Pertussis Antigen P60
3.0
Tetanus toxin fragment C
12.0
HIV gp120
1.25
Tick antigen
1.5
CYTOKINES
TNF
10.0
Interferon alpha
0.4
PROTEASES
Carboxypeptidase B
0.8
ANTIBODIES
Rabbit single chain Fv
0.25
ADVANTAGES OF
EXPRESSION IN P. pastoris
• EUKARYOTE- some post-translational
modification
• MICRO-ORGANISM
– easy to manipulate
– cheap
• YEAST – advanced molecular genetics
• HIGH YIELDS
Molecular Farming
1. A new field where plants and animals are
genetically engineered to produce important
pharmaceuticals, vaccines, and other valuable
compounds.
2. Plants may possibly be used as bioreactors to
mass-produce chemicals that can accumulate
within the cells until they are harvested.
3. Soybeans have been used to produce
monoclonal antibodies with therapeutic value for
the treatment of colon cancer.
Molecular Farming
4.
Plants have been engineered to produce human
antibodies against HIV
5. Pharmaceuticals has begun clinical trials with herpes
antibodies produced in plants.
6. The reasons that using plants may be more cost-effective
than bacteria:
a) Scale-up involves just planting seeds.
b) Proteins are produced in high quantity.
c) Foreign proteins will be biologically active.
d) Foreign proteins stored in seeds are very stable.
e) Contaminating pathogens are not likely to be present.
Molecular Farming
Edible Vaccines
a)
b)
c)
d)
e)
People in developing countries have limited access to many
vaccines.
Making plants that produce vaccines may be useful for
places where refrigeration is limited.
Potatoes have been studied using a portion of the E. coli
enterotoxin in mice and humans.
Other candidates for edible vaccines include banana and
tomato, and alfalfa, corn, and wheat are possible candidates
for use in livestock.
Edible vaccines may lead to the eradication of diseases such
as hepatitis B and polio.
For the last decade, scientists have known how to genetically engineer a plant
to produce a desired protein. The two most common tools used to do this are:
Cut out the selected
region of the plasmid.
Infect the plant with
the agrobacteria and
grow it in a medium.
Agrobacteria have a circular
form of DNA called plasmids.
The plasmids are easily
manipulated because they
naturally
havegene.
two “cut” Grow the plant like
Add the desired
a regular crop.
points where a gene can be
taken out and replaced with
one of the scientist’s choice.
DNA is coated on
microscopically tiny gold
beads that are placed in a
vacuum chamber. The
gene gun then allows
compressed gas to expand,
pushing the beads down
until they hit a filter. The
DNA then flies off of the
beads down into the tissue,
where some will enter a
nucleus and become
incorporated.
Growing plants is much cheaper
than producing vaccines.
The plants that produce the
edible vaccines could be
grown in third world
countries.
Advantages
Agricultural
products
can be
transported
around the
world
relatively
cheaply.
Plants are already
regularly used in
pharmaceuticals, so
there are established
purification
protocols.
Plants can’t host most
human pathogens, so the
vaccines won’t pose
dangers to humans.
Plants are living organisms
that change, so the
continuity of the vaccine
production might not be
guaranteed.
If the vaccines were grown in
fields or on trees, security
would become a big issue.
Disadvantages
The dosage of the vaccines
would be variable. For
example, different sized
bananas would contain
different amounts of
vaccine.
The edible vaccines
could be mistaken
for regular fruits
and consumed in
larger amounts than
might be safe.
Glycosylation
patterns in
plants differ
from those in
humans and
could affect
the
functionality of
the vaccines.
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)
EBNA-1 protein
drives episomal
replication of
ori-P containing
plasmids
integrated Ad5
E1a/E1b fragment
in HEK 293 cells
enhances transcription of CMV
promotor driven
transgene
The cell line is available from ATCC and,
until recently, also from Invitrogen
Why HEK.EBNA Cells?
Advantages
• In comparison to other eukaryotic expression systems
the 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 membranebound, but also intracellular proteins can successfully
be expressed
HEK.EBNA Expression Vectors
HpaI
EcoRV
ScaI
MluI
Am picillin
OriP
pRS5a
ColE1
6372 bps
Bs aM1
DraIII
SV40-EM-Zeocin
BGHpA
CMV
• Basic vector (also
Gateway™ adapted)
• Can be decorated with
N- or C-terminal tags,
heterologous leader
sequences
• Co-expression of e.g.
GFP via IRES element
• Selectable marker for
generation of stable
cell line
SacI
StuI
XhoI
NheI
Commercially available HEK.EBNA vectors:
pREP4 and pCEP4 (Invitrogen)
A Transient Transfection Run…..
25
10
9
Cell density after addition
of 1.4 l transfection mix
20
8
Cell density after addition
of 5 l growth medium
7
15
6
5
10
4
3
5
2
1
0
0
0
20
40
60
80
100
120
time [h]
cell density
product titer
140
160
180
product titer [mg/l]
cell density [ x 10 5 cells/ml]
Cell density in 3.6 volume
prior to transfection
….in Multiparallel Fashion
Cell/Supernatant Harvest and
Cell Lysis
Secreted
product
in supernatant
or
Cell
concentration
Cell
concentrate
Super
natant
Intracellular
product:
Cell concentrate
+ Lysis buffer
Released product
in cleared lysate
Cell debris
Clear
Lysate