Expression of proteins in yeast systems

Jaana ToikkanenVTT Biotechnology

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Expression of proteins in yeast systems

• Introduction to recombinant protein production• Yeast Saccharomyces cerevisiae• Expression of proteins in S. cerevisiae• Secretory pathway of S. cerevisiae• Attempts to enhance production of secreted proteins in

Saccharomyces cerevisiae• Nonconventional yeasts in biotechnology

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Cell fysiology - knowledge and engineeringChanges in fysiology caused by protein overproductionBioprocess design for optimized production and recovery

Gene expressionin the new host

Posttranslationalmodifications

Protein secretionand intracellulartransport

Downstreamprocessing

– Translocation tothe secretorypathway

– Folding andmodifications

– Glycosylation

– The secretorypathway: ER →Golgi → secret.vesicles → PM

– Secretion to thegrowth medium

– Chemicalcharacter ofthe protein

– Proteinengineering

– Recovery &purification

– Regulation ofgene expression

– Molecularbiology methods

Protein production steps

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Gene technology of protein production

General structure of genes

promoter

signal

Gene/ORF

intron

terminator

• Promoters are regulatory units and they may be strong of week• Signal sequences direct the produced protein out of the cell• Introns are found in eukaryotict genes• cDNA produced from the mRNA does not contain introns• Organisms use slightly different codon-preferences

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Introduction to protein production

• Proteins may be intracellular or extracellular

• Intracellular proteins may be produced intracellularly or directed outof the cell by adding a signal sequence or by construction of a fusionprotein with an extracellular protein

• In bacteria, proteins may end in the periplasmic space => formationof inclusion bodies

• Production may be enhanced by the use of fusion protein strategies(+introduction of specific protease sites for cutting of partners later)

• PCR has made it much easier to make the constructions needed

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Introduction to protein production

• Homologous production = production by the gene donating hostorganism

• Heterologous production = production by a different host organism• Classical mutagenesis can be used for increasing homologous

expression• Recombinant production may be facilitated by introducing the gene in

an autonomously replicating plasmids or by integrating it to thegenome

• Copy number of genes in the organism can be increased• The degradation of the product by proteases may be controlled by

e.g. use of protease defective mutants or by changing the sequenceof the protein

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Posttranslational modifications

• Almost all cells (except most prokaryotes) glycosylate their (extracellular)proteins. Glycosylation is species specific

• N-glycosylation occur at Asn in protein sequences Asn-x-Thr/Ser (x notPro). O-glycosylation occur usually at repeated sequences of Ser and Thr

• Many chaperons have been found that help proteins to fold during/aftertranslation

• The signal sequence is cut off before the protein leaves the cell• Some proteins contain pro- and prepro- sequences needed to be cut off

for activity• Some proteins are phosphorylated or modified in other ways

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Recombinant protein biosynthesis

• Optimal conditions for cell growth are typically sub-optimal orincompatible for recombinant protein overproduction

• Protein overproduction:Very demanding for energy (ATP) and reducing power (NADPH)Depends strongly on microbial growth rate

• Understand metabolic changes resulting from proteinoverexpression

• Evaluate product formation at various physiological states• Identify possible bottlenecks• Address such bottlenecks genetically or by optimizing cultivation

conditions

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Cellular responses to secretion stress

SECRETION

SECRETION

ER

NUCLEUSSecretedprotein

Bippdi

BipPDI

tunikamycinDTT

Ca++-ionophors

Sec mutationsHeterologous

protein

PDI

Bip

•unfolded proteinresponse (UPR)=induction of genesinvolved in proteinfolding and removalfrom ER

•attenuation oftranslation (observedonly in mammaliancells)

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What limits protein overexpression?Cellular as well as environmental factors

Biological Chemical Physical

Microbial Strain Media Composition(C, N, P, S, micro-nutrients, surfactants)

Temperature

Gene Dosage,Induction

Dissolved O2, CO2Levels

Gas TransferRates

mRNA Stability Chemical Inducer Sheer Stress

TranslationalCapacity (ribosomalRNA)

pH Cell Morphology(filaments vs. pellets)

Protein Folding,Secretion,Degradation

Growth rate and phase

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Desired properties for protein production host

Genetic methods for strain construction• vectors for replication and integration• selective markers• efficient transformation

Protein production• availability of strong constitutive and inducible promoter• codon usage• posttranslational processing• product stability• GRAS status

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Desired properties for protein production host

Secretion• signal sequence

Physiology• growth conditions• carbon and energy sources

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highhighlowlowCost of growth medium

yesnononogamma-Carboxylation

yesyesyesnoAcylation

yesyesyesnoAcetylation

yesyesyesnoPhosphorylation

yesyesyesnoO-linked glycosylation

complexsimple, no sialicacid

high mannosenoneN-linked glycosylation

proper foldingproper foldingrefolding maybe required

refolding usuallyrequired

Protein folding

Posttranslational modifications

secretion tomedium

secretion tomedium

secretion tomedium

secretion toperiplasm

Extracellular expression

low-moderatelow-highlow-highhighExpression level

complexcomplexminimumminimumComplexity of growth medium

slow (24 h)slow (18-24 h)rapid (90 min)rapid (30 min)Cell growth

Mammalian cellsInsect cellsYeastE. coliCharacteristics

Comparison of expression systems

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Yeast Saccharomyces cerevisiae

• Well known genetics• Well developed genetic methods• A model to other eukaryotes• Full genomic sequence known• About 6000 genes• GRAS• No toxins• Ability to secrete proteins• Capacity for posttranslational

modifications

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Yeast vectors

YIp = yeast integrating plasmid, single copy• for integration into genomic locus

YCp = yeast centromeric plasmid• replicating single copy plasmid• for e.g. complementation studies

YEp = yeast episomal plasmid• replicating multicopy (tens of copies) plasmid• based on yeast 2µ plasmid• for recombinant protein production, multicopy suppression

studies etc.

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Yeast gene technology

Transformation methods:• spheroplast transformation• whole cell transformation using LiAc• electroporation

Selection of transformants:• complementation of auxotrophic mutations

• strain carries a mutation in a given gene (ura3, leu2, trp1, his3)• corresponding wt copy of the gene (URA3, LEU2, TRP1, HIS3)

present in the vector• dominant selection markers

• resistance to G418, cycloheximide, formaldehyde, Cu2+

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Yeast promoters most commonly used forexpression of heterologous genes

Gene Protein Induction/Derepression

SS

ADC1PGK

Alcohol dehydrogenase IPhosphoglyseratekinase

-Fermentable carbon source

--

GAPDH Glyseraldehyde-3-phosphatedehydrogenase

Fermentable carbon source -

PHO5 Acid phosphatase Low PI +

Gal1 GAL10 Galactokinase Galactose -

SUC2 Invertase Low glucose +MFα1 Mating pheromone a MATα +

ADRIII Alcohol dehydrogenase 2 Low glucose -

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MIDDLESHORT

LONG

100

50

20

10

5

2

1

0 0.4 0.8 1.2 1.6 2.0 2.2Cell dry weight (mg/ml)

αα αα-A

myl

ase

units

x 1

0-2

/ mg

cell

dry

wei

ght

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0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0

-0.050 20 40 60 80 100 120

Prod

uctiv

ity (U

/ g.

h) "long""middle""short"

Time (h)

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HIGH MANNOSE

HIGH MANNOSE

COMPLEX

MANNAN

YEAST GLYCOPROTEIN

MAMMALIAN GLYCOPROTEIN

GlcNAc

• Fungal type glycans are composed ofmannose only (S. pombe galactose)

• S. cerevisiae exceptional in makingextensive mannosylation - not in allproteins

• Yeast type glycosylation may affectproperties of the protein product e.g.activity or binding

• α-1,3-Man linkage at the chain terminus isimmunogenic - foreign to mammals

Yeast glycosylationMan

GlcNAc

GalMan

Sialic Acid

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Bacillus αααα-amylase produced in yeast

Y B Y BWestern blot Coomassie

stained

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The fusion strategy for enhanced folding and secretion

Staphylococcus protein A (SPA)Maltose binding protein (MBP)Thioredoxin (DsbA)Glucoamylase (GA)Cellobiohydrolase I (CBHI)

FUSION PROTEINS IN OTHER SECRETORY SYSTEMS

Carrier protein

Escherichia coli

Aspergillus nigerTrichoderma reesei

Host

Yeast MFαααα1p fusion

KEX2

heterologous protein

potential N-glycosylation site

KEX2 KEX2

αααα factor-Lys-Arg-Glu-Ala-Glu-Ala-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-Lys-Arg-

KEX2(yscF)

KEX2(yscF)

KEX2

KEX1(yscαααα)

carrier part

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V

N

TGN

G

ER

EE

P

MM

SV

PEV

?LE

Yeast secretory pathway

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The secretory pathway genes of yeast

YPT7VPS33/SLP1

SEC65SRP54SRP101SSA1-SSA4

V

SEC61, SSH1SSS1SEB1,2SEC62SEC63SEC66/SEC71SEC67/SEC72SEC70

KAR2

SEC16SEC19SEC21SEC26SEC27

SEC12SAR1SEC13SEC23SEC24SEC31

YPT1SLY1SEC17SEC18

SEC22BET1

BET2BET3YKT6BET4SED5USO1SEC7

YPT7NYV1 E

END3END4

VPS21/YPT51VPS45

PEP12VTI1

ERD2SED4SEC20

SEC14 ANP1 ERD1PMR1 MNN9KEX1 VAN1KEX2 VRG4STE14SFT1

VPS1CHC1CLC1

GOS1

VPS15VPS34

SNC1,2SSO1,2SEC1, SEC2, SEC3SEC4, SEC4-GDP

SEC5, SEC6SEC8, SEC9SEC10, SEC15MSO1, DSS4, SMY1SEC17, SEM1, TPM1SEC18, SCD5SEC19, MYO2

G

N

PDI1 EUG1 FKB1 ERN1 SHR3 SEC11

SEC53SEC59

RET1RET2RET3UFE1

CNE1

ER

BOS1

ARF1ARF2SEC21

PTH1/VAM3

ACT1

MPD1

TRE1

VMA22

VMA21

VMA12 Sv

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Ways of increasing protein secretion in yeast

Isolation of super secretory mutants• several isolated• most do not affect secretion• almost all are recessiveFusion of the heterologous protein to a well secreted endogenous protein• prepro α-factor• Hsp 150Deletion of a quality control gene• CNE1 (Calnexin homologue) -> misfolded proteinsOverexpression of er lumenal folding machinery• PDI1 (protein disulfide isomerase)• HAC1Overexpression of components of the secretory machinery• SEB1 (ER translocon component)• SSO1, SSO2 (plasma membrane t-SNARES)Optimization of production conditions• Rich medium• Low temperature• pH

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Nonconventional yeasts in biotechnology

• Schwannimyces occidentalis• Kluyveromyces lactis• Pichia pastoris• Pichia guilliermondii• Pichia methanolica (Pichia pinus MH4)• Hansenula polymorpha (Pichia angusta)• Yarrowia lipolytica• Arxula adeninivorans• Candida maltosa• Trichosporon

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Kluyveromyces lactis

• Can grow on inexpensive medium containing lactose• Well developed genetics• Product yield can be in g/l scale• Commercially used for chymosin production• GRAS status• Ability to secrete high molecular weight proteins• Protease problems with some proteins

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Pichia pastoris

• Methylotrophic yeast that grows to high cell densities• Strong inducible promoters• Expression from integrated plasmid• Requirement of MetOH for induction (toxicity and need for

explosive-safe fermentors)• Production system commercially available from Invitrogen• High production yields• Both intracellular and secreted protein production• Requires precise fermentation conditions• Product stability can be increased by using protease deficient

strains or fusion to other stably expressed protein

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Fryxell, K.B. et al. (1995) Protein Purification 6: 329-336.Weiss, H.M. et al. (1995) FEBS Letters 377: 451-456.

0.050.001

Membrane ProteinsHuman CD38 (soluble portion)Mouse serotonin receptor

Sreekrishna, K. et al. (1989) Biochemistry 28: 4117-4125.Hagenson, M.J. et al. (1989) Enzyme Microbial Technology 11: 650-656.Clare, J.J. et al. (1991) Gene 105: 205-212.Garcia, J.N. et al. (1995) Yeast 11: S589.

10.00.080.450.4

Regulatory ProteinsTumor necrosis factorStreptokinase (active)Mouse epidermal growth factor (EGF)Human IFN-a2b

Clare, J.J. et al. (1991) Bio/Technology 9: 455-460.Scorer, C.A. et al. (1993) Gene 136: 111-119.Scorer, C.A. et al. (1993) Gene 136: 111-119.Rodriguez, M. et al. (1994) J. Biotechnology 33: 135-146.Eldin, P. et al. (1997) J. Immuno. Meth. 20: 67-75.

12.01.250.021.50.25

Antigens and AntibodiesTetanus toxin fragment CHIV-1 gp120 (intracellular)HIV-1 gp120 (secreted)Bm86 tick gut glycoproteinMurine single-chain antibody

Van Nostrand, W.E. et al. (1994) Biochim. et Biophys. Acta 1209: 165-170.Laroche, Y. et al. (1994) Bio/Technology 12: 1119-1124.Brankamp, R.G. et al. (1995) Protein Expression and Purification 6: 813-820.Chang, T. et al. (1997) Biochemistry 36: 7652-7663.

1.01.70.010.1

Proteases and Protease InhibitorsKunitz protease inhibitor (APLP-2)Tick anticoagulant protein (TAP)GhilantentPA Kringle type-2 domain

Despreaux, C.W. and Manning, R.F. (1993) Gene 131: 35-41.Paifer, E. et al. (1994) Yeast 19: 1415-1419.Calera, J.A. et al. (1997) Infection and Immunity 65: 4718-4724.

0.82.52.3

EnzymesD-alanine carboxypeptidaseAlpha amylaseCatalase (Aspergillus fumigatus)

ReferenceExpressionLevel (g / l)Protein Expressed

Selected proteins expressed in Pichia pastoris

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Hansenula polymorpha

• Methylotrophic yeast• Secretes heterologous proteins, in many cases by the leader

peptide of the heterologous protein• Short fermentation times• High level expression can be obtained on glycerol with certain

promoters• Rarely hyperglycosylation of the protein product• Thermotolerant, growth optimum 42°

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Yarrowia lipolytica

• Efficient and precise integrative transformation• Strong regulated and constitutive promoters• Very few compounds may be used as N or C source

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Conclusions

Choices to be made:

Homologous or heterologous production?Genetic engineering needed in heterologous expressionClassical mutagenesis (and genetic engineering) usuallyneeded in homologous expression

Intracellular or extracellular?Wild type versus recombinant protein

Which production host?"Plug and play" a good choice if gene from unknown family

Fermentation system (size)?Down-stream processing?

Keep it as simple as possible!

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If you need help, please contact VTT Biotechnology!

Expression serviceTrichoderma reesei and AspergillusE.coli and BacillusPichia pastoris and SaccharomycesBaculovirus

Fermenters1 - 2000 l for yeast, filamentous fungi and bacteriaUp to 8 l for baculovirus

Down-stream processingSemi-continuous centrifugationDrum filter for fungi, Cross flow filtration, UltrafiltrationChromatography (semi-large scale)Freeze drying and granulation