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