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

Reversetranscriptase

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

SelectableMarker

Promoter

E. coli Promoters

Weickert, et al., 1996

E. coli Expression Vector

SelectableMarker

Promoter

Repressor

E. coli Expression Vector

SelectableMarker

Promoter

TranscriptionalTerminator

SD AUGStop

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 COMMERCIALEPOSSONO ESSERE PRODOTTE IN E. coli CON TECNICHEDNA 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 PROMOTORIFORTI 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 usageTranscript stabilityTranscript secondary structure

Improper secondary, tertiary or quaternary structure formationInefficient or improper disulfide bridge formationInefficient 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

ADPGrpE

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 PROTEINEETEROLOGHE QUESTE VENGONO PRODOTTE COMEPROTEINE 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 oGST

PROMOTOREREGOLABILE

MCS

cDNA di interesse

MBP oGST

TRASFORMAZIONE INBATTERI

INDUZIONE DIESPRESSIONEPROTEINA DI FUSIONE(PROMOTORIREGOLABILI)

SITO DI TAGLIO PER PROTEASI

GST o MBP UTILIZZATE PER PURIFICAZIONE

PROTEINA DIINTERESSE

MBPProteina di fusione

Proteina di fusione purificata

Eluizione

Resina

con legatomaltosio

geneMalE

cDNAdi interesse

Promotore “lac”

pMAL

-

pGEX

tac

•IPTGinduction

•High level expression

GST Foreign gene

GST comes fromSchistosoma mansoni

PURIFICATION OF GST FUSION PROTEINS

PURIFICATION

• EASY

• AFFINITY CHROMATOGRAPHY

PURIFICATIONDETAILS

• 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

proteasedialyse

secondglutathionecolumn

pure foreignpeptide in flowthrough -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

NO

N N N N

HistidineImidazole

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 aaCATENA 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 INSULINARICOMBINANTE 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 UMANOIN 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

T7promoter

pET

foreignantigen

AmpRpIP T7 RNA

polymerase

c1ts= repressor active 28°C, inactive at 37°CpL = 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

tetanustoxoid

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

trcpromoter

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 ofinterest

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 ofinterest

TT

HIS4+

3’ AOX1

3’ AOX1 3’ AOX1 gene ofinterestpAOX1

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

-matingtypesecretion 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. pastoris4. 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 12g per L (INTRACELLULAR)

– amylase 2.5g per L (SECRETED)

CAN WORK ON INDUSTRIAL SCALE

YIELDSPRODUCT 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 FarmingMolecular 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 FarmingMolecular Farming4. 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 FarmingMolecular Farming

Edible VaccinesEdible 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 Potatoes have been studied using a portion of the E. coliE. 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.

AdvantagAdvantageses

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.

DisadvantaDisadvantagesges

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 Ad5E1a/E1b fragment in HEK 293 cells enhances trans-cription of CMVpromotor driventransgene

EBNA-1 protein drives episomal replication ofori-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 systemsthe 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 successfullybe expressed

HEK.EBNA Expression Vectors

pRS5a

6372 bps

HpaI

EcoRV

MluI

SacI

NheI

XhoI

StuI

DraIII

BsaM1

ScaI

OriP

CMV

BGHpASV40-EM-Zeocin

ColE1

Ampicillin

• 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)

A Transient Transfection Run…..

0

5

10

15

20

25

0 20 40 60 80 100 120 140 160 180

time [h]

cell

den

sity

[ x

10

5 c

ells

/ml]

0

1

2

3

4

5

6

7

8

9

10

pro

du

ct t

iter

[m

g/l]

cell density product titer

Cell density in 3.6 volume

prior to transfection

Cell density after additionof 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

Supernatant

Wave bag

Secreted productin supernatant

orCell concentration

Cell debris

Clear Lysate

Intracellular product:

Cell concentrate+ Lysis buffer

Released productin cleared lysate

Wave bag