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)