Espressione di proteine ricombinanti in LIEVITO ......The regulation of the AOX1 gene is therefore...
Transcript of Espressione di proteine ricombinanti in LIEVITO ......The regulation of the AOX1 gene is therefore...
Espressione di proteine ricombinanti in LIEVITO
Saccharomyces cerevisiae & Pichia pastoris
Espressione di proteine ricombinanti in LIEVITO Saccharomyces cerevisiae & Pichia pastoris
Vantaggi del sistema
ü Sono microorganismi eucariotici, evolutivamente più vicini ad organismi complessi.
ü La loro coltivazione in laboratorio è semplice e relativamente economica (assai simile a quella di cellule batteriche).
ü E’ possibile applicare svariate tecniche di ingegneria genetica, sfruttando l’elevata capacità di ricombinazione omologa (gene replacement).
ü Saccharomyces cerevisiae è un organismo modello, molto studiato in termini genomici e proteomici, del quale si conoscono numerosi meccanismi a livello molecolare.
Quali sono le caratteristiche di Saccharomyces cerevisiae?
• Organismo GRAS (Generally Recognized As Safe) • Genetica e fisiologia estremamente ben conosciute
• Cellule isolabili singolarmente e facilmente coltivabili
• Ciclo cellulare sia aploide che diploide
• Metabolismo sia aerobio che anaerobio
• Isolati e sequenziati diversi promotori forti, nonché un plasmidio naturale (2 mm)
• Cellule trasformabili 1) preparazione protoplasti, via chimica o enzimatica 2) trattamento con LiAc 3) elettroporazione
• Secrezione di proteine minima
• Strumento per l’analisi di geni eterologhi • Ridotta complessità genetica
→ Genoma completamente sequenziato → Progetto per l’attribuzione di una funzione alle orphan ORFs
La sua crescita ha un profilo tipico
Le condizioni nutrizionali influenzano la crescita del lievito (andamento della curva, sporulazione)
I terreni di crescita possono essere solidi o liquidi e contengono sali minerali, vitamine, una fonte di azoto e una di carbonio.
Si utilizzano terreni completi e terreni selettivi; esistono infatti numerosi marcatori nutrizionali (e molti composti specifici) che permettono di selezionare i ceppi ingegnerizzati.
Inoltre, la fonte di carbonio utilizzata nella coltivazione del lievito determina l’attivazione di percorsi metabolici specifici.
Esistono fonti di carbonio FERMENTABILI (Glucosio, Galattosio) e RESPIRABILI (Glicerolo, Lattato, Etanolo).
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Glicerolo Galattosio Lattato
COLTIVAZIONE DEL LIEVITO S. cerevisiae
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Long-Term Adaptation
GAL3 è espresso a livello basale in glicerolo, indotto 3-5 volte all’aggiunta di galattosio, strettamente represso in presenza di glucosio.
Nel 1948 (!) venne descritto per la prima volta un mutante incapace di fermentare il galattosio alla stessa velocità del wild-type.
Regolazione dipendente dalla fonte di carbonio
I geni GAL per il metabolismo del Galattosio sono organizzati in un regolone e sono controllati a livello trascrizionale dalla fonte di carbonio
DNA-binding transcriptional activator
repressor
Co-inducer/CYTOPLASM Gal3
Trascrizione geni GAL
Galattosio
I geni GAL per il metabolismo del Galattosio subiscono una regolazione trascrizionale duplice: repressione/derepressione e induzione
Glicerolo Glucosio (± Gal)
L’aggiunta di galattosio fa aumentare di un fattore 1000 la trascrizione del messaggero, fino a raggiungere l’1% degli mRNA totali → un potente e preciso interruttore molecolare.
Elementi che hanno reso possibile l’ingegnerizzazione del lievito:
• isolamento di promotori regolabili (GAL1, PGK glucosio, CUP1 rame);
• isolamento di origini di replicazione → plasmide naturale 2µ, ARS;
• identificazione di markers di selezione → auxotrofie;
• possibilità di ottenere facilmente mutazioni condizionali
→ mutanti termosensibili “ts”;
• elevata frequenza di ricombinazione omologa.
Vettori utilizzabili in lievito
• plasmidi integrativi YIp
• plasmidi replicativi
YEp YRp YCp
• cromosomi artificiali YAC
… e ingegnerizzabili in E.coli, perché vettori navetta
Elementi che hanno reso possibile l’ingegnerizzazione del lievito:
• isolamento di promotori regolabili (GAL1, PGK glucosio, CUP1 rame);
• isolamento di origini di replicazione → plasmide naturale 2µ, ARS;
• identificazione di markers di selezione → auxotrofie;
• possibilità di ottenere facilmente mutazioni condizionali
→ mutanti termosensibili “ts”;
• elevata frequenza di ricombinazione omologa.
Marcatori di selezione: come sono stati originariamente isolati?
Per complementazione in E.coli.
Transform Leu- E.coli Plate onto medium lacking leucine
Marcatori di selezione usati più frequentemente
Elementi che hanno reso possibile l’ingegnerizzazione del lievito:
• isolamento di promotori regolabili (GAL1, PGK glucosio, CUP1 rame);
• isolamento di origini di replicazione → plasmide naturale 2µ, ARS;
• identificazione di markers di selezione → auxotrofie;
• possibilità di ottenere facilmente mutazioni condizionali
→ mutanti termosensibili “ts”;
• elevata frequenza di ricombinazione omologa.
“Gene targeting” mediante ricombinazione omologa
Knock-Out del gene URA3 (knock-in del gene YFG)
Knock-Out del gene YFG (knock-in del gene URA3)
URA3::YFG
Left segment of URA3
Right segment of
URA3
YFG::URA3
Right segment of YFG
Left segment of
YFG
ESPRESSIONE DI PROTEINE RICOMBINANTI IN Saccharomyces cerevisiae MEDIANTE SISTEMI GAL
Ponendo una sequenza codificante sotto il controllo del
promotore GAL1, la sua trascrizione viene fortemente indotta in terreno contenente
Galattosio e può essere immediatamente repressa in
presenza di Glucosio
Ciò permette un controllo dell’espressione basato sulla variazione delle condizioni di
crescita
Nel 1984 viene prodotto in lievito il primo vaccino ricombinante costituito da una subunità del capside del virusdell’epatite B. I tentativi di produrlo in E. coli erano precedentemente falliti.
La proteina ricombinante è assai simile a quella naturale e conserva anche la capacità di formare aggregati immunogenici simili a quelli trovati in pazienti infetti.
……
Proteine ricombinanti
prodotte in S. cerevisiae
Problemi possibili per l’espressione in questo sistema:
• perdita del plasmide
• iperglicosilazione di alcune proteine (v. oltre)
• secrezione di proteine limitata
Improved production strains of yeast S.cerevisiae…
Altering the membrane lipid content …
Pichia pastoris
Why is Pichia pastoris advantageous for industrial applications?
• simplicity of the techniques
• capacity to produce large (intra- or extra-cellular) quantities of protein
• possibility of post-translational modifications
• various commercialized systems
• easy to move from the lab- to the fermenter-scale
TASSO NOMIA: Euka ryota , Fung i, Asc omyc ota , Sa c c ha romyc o tina ,Sa c c ha romyc e te s,Sa c c ha romyc e ta le s, Sa c c ha romyc e ta c e a e , Pic hia
Expression of recombinant proteins in Pichia pastoris
Pichia pastoris is a methylotrophic yeast, capable of using methanol as the sole carbon source.
P.pastoris is a yeast capable of expressing the recombinant protein at levels 10-100 times higher than in S. cerevisiae.
Its hyper-glycosilation activity is lower than that of S.cerevisiae and thus its secretory properties are greater.
Some of the genetic engineering strategies used in S.cerevisiae are similar to those employable with Pichia.
Sequencing of the genome completed and published, although relatively late with respect to other species ( ).
Protein-coding genes were automatically predicted using EuGene15... manually curated for functional annotation, accurate translational start-and-stop assignment, and intron location. This resulted in a 5,313 protein-coding gene set of which 3,997 (75.2%) have at least one homolog in the National Center for Biotechnology Information protein database. The protein-coding genes occupy 80% of the genome sequence.
To be used, the methanol must first be oxidized to formaldehyde in peroxisomes by the enzyme alcohol oxidase.
FORMALDEIDE
METANOLO O2
H2O2
alcohol oxidase
alcohol oxidase has low affinity for O2 and, when growing in methanol, the yeast cell increases the production of the enzyme.
Pichia has two alcohol oxidase genes (AOX1, AOX2): AOX1 supports most of the enzymatic cellular activity.
The regulation of the AOX1 gene is therefore used for the production of recombinant proteins in Pichia pastoris.
In methanol, the AOX1 protein represents about 30% of the soluble protein and its mRNA 5% of the cell messengers.
The loss of the gene AOX1 (MutS) confers to the cell a distinctive phenotype, i.e. growth slowed in methanol.
In Pichia pastoris, The gene AOX1 undergoes an adjiustement similar to that of genes GAL in S.cerevisiae: repression / derepression and induction.
Transcription of gene AOX1
Methanolo Glycerol Glucose (± Methanol)
Vectors Promoters
– AOX1 (alcohol oxidase) • Strong promoter • Strongly inducible by methanol • Repressed by D-glucose
– GAP (glyceraldehyde 3-phospate dehydrogenase)
• Strong constitutive promoter • High transcription in D-glucose, • Moderate transcription in glycerol • Low transcription in methanol
Markers ARG4, URA3, HIS4, Sh ble (gene from Streptoalloteicus hindustanus)
Secretion signals α-MF (S.cerevisiae mating factor a), PHO1 (P.pastoris acid phosphatase)
1) Choice of the expression system
INTRACELLULAR system
SECRETION system
Pichia secretes few native proteins, so that the recombinant one can represent the most abundant specie in the culture medium
In the absence of a native signal, the prepropeptide of α-factor of S. cerevisiae is one of the most efficient secretion signals
The peptide is removed by specific proteases prior to secretion (Kex2, Ste13)
recombinant protein with epitopes 6XHis and c-myc at the C-terminal
Expression of recombinant proteins in Pichia pastoris
1) Choice of the expression system
Plasmid for intracellular expression Plasmid for secretion
2) Construction of the specific vector
Expression of recombinant proteins in Pichia pastoris
pPICZα-A Construction of the specific vector
ORF at 5’ (α-factor)
ORFs at 3’ (c-myc – 6xHis)
Kex2 E Ste13 sites (removal of α-factor)
Control by sequencing of the correct insertion of the DNA fragment and of the absence of point mutations or premature STOP codons
1) Choice of the expression system
2) Construction of the specific vector
Expression of recombinant proteins in Pichia pastoris
3) Engineering of Pichia pastoris
A single homologous recombination event allows the integration of the plasmid into the genome
The presence of the marker Zeocin allows selection of the engineered strains
Strain transformation
1) Choice of the expression system
2) Construction of the specific vector
Expression of recombinant proteins in Pichia pastoris
The expression vectors can be cut in such a way as to allow only the integration of the expression cassette and marker gene flanked by sequences 5 'and 3' of the AOX1 gene, replacing the AOX1 gene (knocked-out)
The result is a Muts phenotype that can be identified by plating the cells on methanol → slow growth
Expression of recombinant proteins in Pichia pastoris
4) Control analysis
sequencing of the junction (control of integration)
analytic PCR on genomic DNA to detect possible multiple integrations
3) Engineering of Pichia pastoris
1) Choice of the expression system
2) Construction of the specific vector
Expression of recombinant proteins in Pichia pastoris
Control analysis of the engineered Pichia pastoris strains
The engineering can alter the function of the gene AOX1 of Pichia (phenotype MutS).
It is therefore necessary to carry out the analysis of the phenotype of the strains obtained (growth in methanol).
Sometimes, it is advantageous to use a MutS strain, or a strain mutated in genes encoding proteases.
The high frequency of homologous recombination typically leads to a high number of transformants (> 50)
Moreover, it can be advantageous to use a strain with multiple integrations (in this case, it is useful to use vectors with the gene conferring resistance to kanamycin or to zeocin).
5) Choice of the best engineered strains
Selection of strains by growth on medium containing methanol.
Small-scale cultures and protein analysis of SDS / PAGE and Western blotting.
4) Control analysis
3) Engineering of Pichia pastoris
1) Choice of the expression system
2) Construction of the specific vector
Expression of recombinant proteins in Pichia pastoris
6) Preparation of large scale cultures and protein purifiction
4) Contol analysis
3) Engineering of Pichia pastoris
1) Choice of the expression system
2) Construction of the specific vector
5) Choice of the best engineered strains
- Flasks with liquid media - FERMENTERS (high density cultures)
• Control of nutrients • pH • Aeration
Simply to change the scale of production from flask to fermenter → the yield of the protein can increase significantly ( > 400g/l wet weight; DO600> 500u/ml )
Expression of recombinant proteins in Pichia pastoris
• High scale production in a fermenter
– The transformant selected for the fermentation is initially grown in rich medium with glycerol as the carbon source
• accumulation of biomass • repressed expression
– Glycerol is then added in limited amounts as long as the culture does not reach the desired level of biomass → > cell viability, more rapid induction, > yield of recombinant protein.
– Finally, the administration of methanol to induce the expression can be started.
– N.B .: control [glycerol], [ethanol], [acetate].
Expression of recombinant proteins in Pichia pastoris
Overall, the codon usage is similar to the one for S. cerevisiae. … The codon optimization of the gene of interest and its eventual fusion partners often results in higher protein expression levels.
The commonly used methanol-inducible promoters in P. pastoris—the alcohol oxidase I promoter and the formaldehyde dehydrogenase promoter—drive the production of enzymes needed for methanol assimilation and therefore produce extremely high levels of these transcripts upon switching the carbon source to methanol. The genome sequence has allowed identification of all genes coding for enzymes involved in methanol assimilation and their promoter, which can now be studied for their suitability for transgene expression in P. pastoris.
the results of several articles are taken under review and compared : • in the passage from the flask to the fermenter, the yield of recombinant
protein expressed under the control of AOX1 promoter is not always greater than that obtainable with the GAP promoter;
• glucose, glycerol and oleic acid, as carbon sources are substrates cheaper than methanol and are more easily disposable;
• you can get an increase in the yield , although significant, but not necessarily proportional, by increasing the number of copies of the gene (eg. TNF, 20 copies of the gene → yield increased by 200 times; 19 copies of mEGF → 13 times; 8 copies of HBsAg → 11 times).
NB: unfolding triggers UPR and ERAD pathways !!
• glyco-engineered strains
More than 50% of the total proteins are glycoproteins; it is estimated that 1-2% of the genome encodes genes involved in glycosylation or metabolism of the glycan chains
N-glycosylation: the chain is covalently linked to ASP consensus Asp-X-Ser / Thr (where X is any aa ≠ Pro)
O-glycosylation: Ser or Thr, consensus sequences not yet identified; in the Golgi
C-mannosylation: C2-alpha-mannosyltryptophan [(C2-Man-) Trp]
Phospho-glycosylation: Ser or Thr, via phosphodiester bond
Glycosylation in cells plays a role in:
• Assumption of the correct folding
• stability of the protein
• adhesion between different cells and between identical cells (in tissues)
• internalisation of viruses
• recognition and response to external agents (NB: eg. Leukocytes expose the membrane molecules CAM = cell adesion molecules, extensively glycosylated, which play a key role in inflammatory and immune responses, or glycoproteins surface antigens of red blood cells AB )
Berger, Kaup and Blanchard. Protein Glycosylation and Its Impact on Biotechnology. Adv Biochem Engin/Biotechnol, vol. 127 (2012)165-185
Berger, Kaup and Blanchard. Protein Glycosylation and Its Impact on Biotechnology. Adv Biochem Engin/Biotechnol, vol. 127 (2012)165-185
Berger, Kaup and Blanchard. Protein Glycosylation and Its Impact on Biotechnology. Adv Biochem Engin/Biotechnol, vol. 127 (2012)165-185
3 Glu 9 Man
2 GlcNAc
In terms of N-glycosylation, P. pastoris modify proteins with a range of heterogenous high mannose glycans, which introduce a large amount of heterogeneity in the protein (reducing downstream processing efficiency and complicating product characterization)… To overcome the difficulties, strains have been developed with an entirely re-engineered glycosylation pathway to produce human IgG–type N-glycans (N-glycosylation humanization technology).
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β-1,2-N-acetylglucosaminyltransferase
mannosidase
galactosyltransferase
sialilyltransferase
S.cerevisiae
ü More possibilities for genetic manipulation
ü Very wide choice of resources and tools (strains, vectors, growth media, bioinformatics tools)
ü Comprehensive understanding of the structure è almost unlimited literature
P.pastoris
ü Higher protein yield ü reduced margins of manipulation ü strictly defined growth conditions
ü NB: if the expression is not simple and immediate, process optimization can be very laborious
ü limited literature ü huge interest for industrial
developments
http://www.pichia.com/welcome/
For studying:
• MOLECULAR BIOTECHNOLOGY Glick, Pasternak, Patten – 4th edition – chapter 7 • Ahmad, Hirz, Pichler and Schwab. Protein expression in Pichia pastoris:recent
achievements and perpectives for heterologous protein production. Appl Microbiol Biotechnol (2014) 98: 5301-5317
• Berger, Kaup and Blanchard. Protein Glycosylation and Its Impact on Biotechnology. Adv Biochem Engin/Biotechnol, vol. 127 (2012) 165-185
To explore the topic:
• Puxbaum, Mattanovich and Gasser. Quo vadis? The challenges of recombinant protein folding and secretion in Pichia pastoris. Appl Microbiol Biotechnol (2015) 99:2925–2938
• Spohner, Müller, Quitmann and Czermak. Expression of enzymes in food and feed industry with Pichia pastoris. Journal of Biotechnology (2015) 202: 118-134 (in the second part of the review real examples are presented of enzymes produced by adapting, case by case, strategies and condition for achieving over-expression)