Purificazione di proteine umane da animali
description
Transcript of Purificazione di proteine umane da animali
![Page 1: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/1.jpg)
Purificazione di proteine umane da animali
• Basse rese• Difficili da purificare• Costoso• Possibilita’ di malattie
![Page 2: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/2.jpg)
How can we synthesise human proteins?
• Use bacterial cells• Human gene lacks
• Bacterial promoter• Bacterial terminator• Bacterial ribosome binding site
• Cannot deal with introns
![Page 3: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/3.jpg)
Dealing with introns
DNA
RNA
Protein
RNA
DNA
Reversetranscriptase
![Page 4: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/4.jpg)
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
![Page 5: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/5.jpg)
E. coli Expression Vector
SelectableMarker
Promoter
![Page 6: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/6.jpg)
E. coli Promoters
![Page 7: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/7.jpg)
Weickert, et al., 1996
![Page 8: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/8.jpg)
E. coli Expression Vector
SelectableMarker
Promoter
Repressor
E. coli Expression Vector
SelectableMarker
Promoter
TranscriptionalTerminator
SD AUGStop
Ori
![Page 9: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/9.jpg)
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?
![Page 10: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/10.jpg)
Codon Frequency in E. coli
![Page 11: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/11.jpg)
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?
![Page 12: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/12.jpg)
Expression of Fusion Proteins
• Ease of detection
• Increase solubility
• Increase stability
• Increase expression
• Ease of purification
![Page 13: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/13.jpg)
Examples of Fusions/Tags
• Hexahistidine-tag• GST• MBP• CBP/Intein• Arg-tag• S-tag
• Ni affinity• GSH• Amylose• Chitin• Ion-Exchange• RNAse
![Page 14: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/14.jpg)
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
![Page 15: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/15.jpg)
Improving Protein Stability
• Protease inhibitors• Protease-minus host• Periplasmic expression• Growth temperature• Combined approach
![Page 16: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/16.jpg)
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
![Page 17: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/17.jpg)
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
![Page 18: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/18.jpg)
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
![Page 19: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/19.jpg)
Folding chaperones in de novo folding
Aggregate
3' 5'
K
TF
J
Native
K
ADP
GrpE
J
GroEL
GroES
ATP
ATP
ADP
ATP
ADPGrpE
![Page 20: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/20.jpg)
GroEL-GroES co-expression and low temperatures improve leptin folding
![Page 21: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/21.jpg)
However, this strategy does not always work
![Page 22: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/22.jpg)
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
![Page 23: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/23.jpg)
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
![Page 24: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/24.jpg)
MBPProteina di fusione
Proteina di fusione purificata
Eluizione
Resina
con legatomaltosio
geneMalE
cDNAdi interesse
Promotore “lac”
pMAL
-
![Page 25: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/25.jpg)
pGEX
tac
•IPTGinduction
•High level expression
GST Foreign gene
GST comes fromSchistosoma mansoni
![Page 26: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/26.jpg)
PURIFICATION OF GST FUSION PROTEINS
![Page 27: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/27.jpg)
PURIFICATION
• EASY
• AFFINITY CHROMATOGRAPHY
![Page 28: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/28.jpg)
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
![Page 29: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/29.jpg)
GLUTATHIONE SEPHAROSE
glutathione
SEPHAROSE
![Page 30: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/30.jpg)
FUSION PROTEIN
GST
FOREIGN PEPTIDE
![Page 31: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/31.jpg)
FUSION PROTEIN BOUND TO GLUTATHIONE SEPHAROSE
glutathione
GST
FOREIGN PEPTIDE
SEPHAROSE
![Page 32: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/32.jpg)
PURIFICATION
• WASH COLUMN EXTENSIVELY
• ELUTE WITH REDUCED GLUTATHIONE
• RESULTS IN PURE GST FUSION PROTEIN
![Page 33: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/33.jpg)
COMPETITIVE ELUTION WITH GLUTATHIONE
SEPHAROSE
![Page 34: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/34.jpg)
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
![Page 35: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/35.jpg)
pQE VECTORS (Qia Express)
• Hex-histidine tag system
• Produce peptides with 6 histidines fused to N or C terminus
• Allows Nickel Chelate Affinity Chromatography
![Page 36: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/36.jpg)
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
![Page 37: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/37.jpg)
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
![Page 38: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/38.jpg)
pQE VECTORS (Qia Express)
• Elution– competitive with imidazole
NO
N N N N
HistidineImidazole
![Page 39: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/39.jpg)
pQE VECTORS (Qia Express)
• Removal of His tag?– not necessary usually– many hundreds of proteins purified with no
effect on structure– not immunogenic
![Page 40: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/40.jpg)
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
![Page 41: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/41.jpg)
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
![Page 42: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/42.jpg)
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
![Page 43: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/43.jpg)
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)
![Page 44: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/44.jpg)
PRODUZIONE DI GH RICOMIBINATE IN BATTERI
![Page 45: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/45.jpg)
SALMONELLA
• Expression host
• Live vaccine delivery
![Page 46: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/46.jpg)
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
![Page 47: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/47.jpg)
SALMONELLA
• Recognises E.coli promoters and origins of replication– therefore existing vectors can function
• Several ways of attenuating Salmonella have been discovered
![Page 48: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/48.jpg)
EXPRESSION SYSTEMS
MOST USE PLASMIDS– PROBLEMS
• INSTABILITY
• TOXICITY
• pIP-pET DUAL PLASMID
• NirB-ANAEROBIC INDUCIBLE
• BALANCED LETHAL
![Page 49: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/49.jpg)
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
![Page 50: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/50.jpg)
pTECH VECTORS
• THESE USE THE NIRB PROMOTER
• NIRB ENCODES NADH-DEPENDENT NITRITE REDUCTASE
• NIRB INDUCED IN ANAEROBIC CONDITIONS eg GUT & TISSUES
![Page 51: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/51.jpg)
pTECH VECTORS
NirB promoter
pTECH
GST
AmpR
tetanustoxoid
Khan made this vector Oral immunisation, single dose in mice-protected against Salmonella Tetanus toxin
![Page 52: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/52.jpg)
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
![Page 53: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/53.jpg)
Balanced Lethal
trcpromoter
pYA292
foreign gene
asd
asd complements asd host & is thus stable
![Page 54: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/54.jpg)
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
![Page 55: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/55.jpg)
EXPRESSION IN PICHIA PASTORIS
![Page 56: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/56.jpg)
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
![Page 57: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/57.jpg)
STEP1• CLONE GENE OF INTEREST INTO
SHUTTLE VECTOR DOWNSTREAM OF AOX1 PROMOTER IN E. coli
AOX1 promoter
gene ofinterest
TT
HIS4+
3’ AOX1
![Page 58: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/58.jpg)
STEP2• TRANSFORM HIS- PICHIA PASTORIS YEAST
WITH PLASMID. SELECT FOR HIS+ STABLE INTEGRANTS DISRUPTED IN THE AOX1 LOCUS
![Page 59: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/59.jpg)
STEP2
AOX1 promoter
gene ofinterest
TT
HIS4+
3’ AOX1
3’ AOX1 3’ AOX1 gene ofinterestpAOX1
TT
INTEGRATION
P.pastoris chromosome
![Page 60: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/60.jpg)
• Pichia pastoris production of single-chain antibody fragments (scFv)
• A CASE STUDY
1. PLACE scFv cDNA in vector pPIC9K
![Page 61: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/61.jpg)
pPIC9K
pAOX1 scFv cDNA His 6 tag
-matingtypesecretion signal
PLACE scFv cDNA in vector pPIC9K
ALL RECOMBINANT STEPS DONE IN E.coli
![Page 62: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/62.jpg)
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
![Page 63: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/63.jpg)
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
![Page 64: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/64.jpg)
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
![Page 65: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/65.jpg)
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
![Page 66: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/66.jpg)
ADVANTAGES OF EXPRESSION IN P. pastoris
• EUKARYOTE- some post-translational modification
• MICRO-ORGANISM– easy to manipulate – cheap
• YEAST – advanced molecular genetics• HIGH YIELDS
![Page 67: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/67.jpg)
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.
![Page 68: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/68.jpg)
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.
![Page 69: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/69.jpg)
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.
![Page 70: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/70.jpg)
![Page 71: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/71.jpg)
![Page 72: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/72.jpg)
![Page 73: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/73.jpg)
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.
![Page 74: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/74.jpg)
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.
![Page 75: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/75.jpg)
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.
![Page 76: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/76.jpg)
![Page 77: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/77.jpg)
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
![Page 78: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/78.jpg)
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
![Page 79: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/79.jpg)
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)
![Page 80: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/80.jpg)
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
![Page 81: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/81.jpg)
….in Multiparallel Fashion
![Page 82: Purificazione di proteine umane da animali](https://reader036.fdocuments.us/reader036/viewer/2022070418/56815946550346895dc68191/html5/thumbnails/82.jpg)
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