Purificazione di proteine umane da animali
• 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
DNA
Reverse
transcriptase
Protein Expression in E. coli
• Inexpensive
• Easy to manipulate
• Well characterized
• Grows quickly
• rProtein up to 50%
total protein
• Post-transcriptional modification
• Post-translational modification
• Poor folding
• Proteolysis
• N-terminal Methionine
• Complicated purification
• Lack of efficient secretion
• Possible toxicity
Advantages and Disadvantages
E. coli Expression Vector
Selectable Marker
Promoter
E. coli Promoters
Weickert, et al., 1996
E. coli Expression Vector
Selectable Marker
Promoter Repressor
E. coli Expression Vector
Selectable Marker
Promoter
Transcriptional Terminator SD AUG
Stop
Ori
Optimizing Expression
• Examine codon usage
– Decrease message stability
– Premature termination of transcription – Premature termination of translation
– Frameshifts, deletions, and misincorporation
What if expression is low?
Codon Frequency in E. coli
Optimizing Expression
• Combined approach
• Examine codon usage
• Minimize GC at 5’
• Add terminator
• Add fusion and/or tags
• Growth conditions
What if expression is low?
Expression of Fusion Proteins
• Ease of detection
• Increase solubility
• Increase stability
• Increase expression
• Ease of purification
Examples of Fusions/Tags
• Hexahistidine-tag
• GST
• MBP
• CBP/Intein
• Arg-tag
• S-tag
• Ni affinity
• GSH
• Amylose
• Chitin
• Ion-Exchange
• RNAse
Insoluble Proteins
• 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
• 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
Promoter choice and design
Inefficient transcription No or little protein synthesized
Codon usage Transcript stability
Transcript secondary structure
Improper secondary, tertiary or quaternary structure formation Inefficient or improper disulfide bridge formation
Inefficient isomerization of peptidyl-prolyl bonds Inefficient translation No or little protein synthesized
Inefficient folding (cytoplasmic or periplasmic)
Inefficient membrane insertion/translocation Toxicity Cell death
Aggregation or degradation
Aggregation or degradation
Folding chaperones in de novo folding
Aggregate
3' 5'
K TF
J
Native
K
ADP
GrpE
J
GroEL GroES
ATP
ATP
ADP ATP
ADP
GrpE
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
MCS
cDNA di interesse
MBP o GST
PROMOTORE REGOLABILE
MCS
cDNA di interesse
MBP o GST
TRASFORMAZIONE IN BATTERI
INDUZIONE DI ESPRESSIONE PROTEINA DI FUSIONE
(PROMOTORI REGOLABILI)
SITO DI TAGLIO PER PROTEASI GST o MBP
UTILIZZATE PER PURIFICAZIONE
PROTEINA DI INTERESSE
MBP
Proteina di fusioneProteina di fusione purificata
Eluizione
Resina con legato maltosio
gene MalE
cDNA
di interesse Promotore
“lac”
pMAL
-
pGEX
tac
•IPTG induction
•High level expression
GST Foreign gene
GST comes from Schistosoma
mansoni
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
GST
FOREIGN PEPTIDE
FUSION PROTEIN BOUND TO GLUTATHIONE SEPHAROSE
glutathione GST
FOREIGN PEPTIDE
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
protease dialyse
second
glutathione column
pure foreign peptide in flow through -
GST sticks
+ GST
foreign peptide pure fusion protein
+ glutathione
pure fusion
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
– 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 non- conformation dependent manner
– therefore resists strong denaturants eg 6M
guanidium HCl
pQE VECTORS (Qia Express)
• Elution
– competitive with imidazole
N O
N N N N
Histidine 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
ESONE 1 ESONE 2
CATENA A 30 aa
CATENA B 21 aa Unite da ponti S-S
PREPROINSULINA
PROINSULINA
INSULINA
PEPTIDE SEGNALE FORMA S-S
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
T7
promoter
pET foreign antigen
AmpR
pIP T7 RNA
polymerase
c1ts= repressor active 28°C, inactive at 37°C pL = left promoter
c1ts
pL
kanR
pTECH VECTORS
• THESE USE THE NIRB PROMOTER
• NIRB ENCODES NADH-DEPENDENT NITRITE REDUCTASE
• NIRB INDUCED IN ANAEROBIC
CONDITIONS eg GUT & TISSUES
pTECH VECTORS
NirB promoter
pTECH GST
AmpR tetanus toxoid
Khan made this vector 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
trc
promoter
pYA292
foreign gene
asd
asd complements asd host & is thus stable
Heterologous Expression in Yeast
• 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
AOX1 promoter gene of
interest TT
HIS4+
3’ AOX1
STEP2
• TRANSFORM HIS
-PICHIA PASTORIS YEAST
WITH PLASMID. SELECT FOR HIS+ STABLE
INTEGRANTS DISRUPTED IN THE AOX1 LOCUS
STEP2
AOX1 promoter gene of
interest
TT HIS4+
3’ AOX1
3’ AOX1 gene of 3’ AOX1 interest
pAOX1
TT
INTEGRATION
P.pastoris chromosome
• Pichia pastoris production of single-chain antibody fragments (scFv)
• A CASE STUDY
1. PLACE scFv cDNA in vector pPIC9K
pPIC9K
pAOX1
scFv cDNA His 6 tag
-mating type secretion signal
PLACE scFv cDNA in vector pPIC9K
ALL RECOMBINANT STEPS DONE IN E.coli
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 @ 30
oC 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 12g per L (INTRACELLULAR)
– amylase 2.5g per L (SECRETED)
CAN WORK ON INDUSTRIAL SCALE
YIELDS
PRODUCT YIELD g per L
ENZYMES
Invertase 2.3
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 Molecular Farming
1. 1. A new field where plants and animals are A new field where plants and animals are genetically engineered to produce important genetically engineered to produce important pharmaceuticals, vaccines, and other valuable pharmaceuticals, vaccines, and other valuable compounds.
compounds.
2. 2. Plants may possibly be used as bioreactors to Plants may possibly be used as bioreactors to mass-produce chemicals that can accumulate mass-produce chemicals that can accumulate within the cells until they are harvested.
within the cells until they are harvested.
3. 3. Soybeans have been used to produce Soybeans have been used to produce
monoclonal antibodies with therapeutic value for monoclonal antibodies with therapeutic value for the treatment of colon cancer.
the treatment of colon cancer.
Molecular Farming 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 Molecular Farming
Edible Vaccines Edible Vaccines
a)a) People in developing countries have limited access to many People in developing countries have limited access to many vaccines.
vaccines.
b)b) Making plants that produce vaccines may be useful for Making plants that produce vaccines may be useful for places where refrigeration is limited.
places where refrigeration is limited.
c)c) Potatoes have been studied using a portion of the E. coliPotatoes have been studied using a portion of the E. coli enterotoxin in mice and humans.
enterotoxin in mice and humans.
d)d) Other candidates for edible vaccines include banana and Other candidates for edible vaccines include banana and tomato, and alfalfa, corn, and wheat are possible candidates tomato, and alfalfa, corn, and wheat are possible candidates for use in livestock.
for use in livestock.
e)e) Edible vaccines may lead to the eradication of diseases such Edible vaccines may lead to the eradication of diseases such as hepatitis B and polio.
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:
Agrobacteria have a circular form of DNA called plasmids.
The plasmids are easily manipulated because they naturally have two “cut”
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.
Cut out the selected region of the plasmid.
Add the desired gene. Grow the plant like a regular crop.
Infect the plant with the agrobacteria and grow it in a medium.
Advantag Advantag es
es
The plants that produce the edible vaccines could be grown in third world countries.
Growing plants is much cheaper than producing vaccines.
Plants are already regularly used in pharmaceuticals, so there are established purification
protocols.
Agricultural products can be
transported around the world
relatively cheaply.
Plants can’t host most human pathogens, so the vaccines won’t pose
dangers to humans.
Disadvanta Disadvanta ges ges
Plants are living organisms that change, so the
continuity of the vaccine production might not be guaranteed.
Glycosylation patterns in plants differ from those in humans and could affect the
functionality of the
vaccines.
If the vaccines were grown in fields or on trees, security would become a big issue.
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.
Why HEK.EBNA Cells? The Principle
integrated Ad5 E1a/E1b fragment in HEK 293 cells enhances trans- cription of CMV promotor driven transgene
EBNA-1 protein drives episomal replication of ori-P containing plasmids
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 systems the HEK.EBNA Expression System is rapid:
from gene to protein in 4-6 weeks
• It can be applied to generate stable cell lines (pools/ clones) and in transient mode on
small and large scale
• The cells can be grown adherently and in serum-free suspension culture
• In transient mode not only secreted and membrane-
bound, but also intracellular proteins can successfully
be expressed
HEK.EBNA Expression Vectors
pRS5a
6372 bps
HpaI
EcoRV
MluI
SacI
NheI XhoI StuI
DraIII BsaM1
ScaI OriP
CMV BGHpA
SV40-EM-Zeocin ColE1
Ampicillin
• 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
Commercially available HEK.EBNA vectors:
pREP4 and pCEP4 (Invitrogen)
A Transient Transfection Run…..
0 5 10 15 20 25
0 20 40 60 80 100 120 140 160 180
time [h]
cell density [ x 105 cells/ml]
0 1 2 3 4 5 6 7 8 9 10
product titer [mg/l]
cell density product titer
Cell density in 3.6 volume prior to transfection
Cell density after addition of 1.4 l transfection mix
Cell density after addition of 5 l growth medium
….in Multiparallel Fashion
Cell/Supernatant Harvest and Cell Lysis
Cell concentrat
e
Super natant Wave
bag
Secreted product in
supernatant or
Cell
concentratio n
Cell debris
Clear Lysate
Intracellular product:
Cell
concentrate + Lysis buffer Released
product in cleared lysate
Wave bag