heterologous protein expression in yeast coho7e - green, core and ha malcolm stratford & hazel...
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Heterologous Protein Expression Heterologous Protein Expression in Yeastin Yeast
CoHo7e - Green, Core and HACoHo7e - Green, Core and HA
Malcolm Stratford & Hazel SteelsMalcolm Stratford & Hazel Steels
MOLOGICMOLOGIC
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ContentsContents
Introduction Introduction Core+GFP expression in Core+GFP expression in Pichia pastorisPichia pastoris SonicationSonication CTABCTAB Protein gelsProtein gels Protein measurementProtein measurement Antibody detectionAntibody detection
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Yeast expression systemYeast expression system
Pichia pastorisPichia pastoris, integrating vector, , integrating vector, zeocin selection, AOX promoter zeocin selection, AOX promoter induced by methanolinduced by methanol
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CoHo7e-GFP formation in CoHo7e-GFP formation in P. pastorisP. pastoris
Induction by methanol, optimised at Induction by methanol, optimised at 0.7% daily 0.7% daily
Optimal induction in highly aerated Optimal induction in highly aerated cultureculture
Cultures progressively turn green, Cultures progressively turn green, after 3 days, progressively increasing after 3 days, progressively increasing to 10 daysto 10 days
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CoHo7e-GFP in P. pastoris, 5 days induction
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Green – Physical ExtractionGreen – Physical Extraction
Grinding with glass beads yields a Grinding with glass beads yields a green slurrygreen slurry
Hard centrifugation showed almost Hard centrifugation showed almost all green fluorescence in the pelleted all green fluorescence in the pelleted fractionfraction
However, there was an intense green However, there was an intense green fluorescent layer at the top of the fluorescent layer at the top of the pelletpellet
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Green SlurryGreen Slurry
Contains yeast cell debris and ~ 10% GFP Contains yeast cell debris and ~ 10% GFP in solid particlesin solid particles
Green particles appear damaged by Green particles appear damaged by grinding but remain substantially intactgrinding but remain substantially intact
No evidence of membranes around No evidence of membranes around particles – look like solid protein inclusion particles – look like solid protein inclusion bodies. bodies.
Without fluorescence, green particles Without fluorescence, green particles indistinguishable from other debrisindistinguishable from other debris
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Tris/pH matrixTris/pH matrix
Water extraction
100m
M
80m
M
60m
M
50m
M
40m
M
30m
M
25m
M
20m
M
15m
M
10m
M
5mM
2mM
pH 6.0
pH 6.5
pH 7.0
pH 7.5
pH 8.0
pH 8.5
% soluble, Tris/pH matrix
85-100
70-85
55-70
40-55
25-40
10-25
-5-10
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pH adjustment without bufferspH adjustment without buffers
Water extraction
0.00
20.00
40.00
60.00
80.00
100.00
120.00
pH 6.0 pH 6.5 pH 7 pH 7.5 pH 8 pH 8.5 pH 9
% S
olu
ble
NaOH
KOH
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Extracting CoHo7e-GreenExtracting CoHo7e-Green
Most ~ 60% - 80% Green is made Most ~ 60% - 80% Green is made soluble by raising the pH to 8.0 in soluble by raising the pH to 8.0 in low ionic strength. low ionic strength.
Can we use sonication or CTAB to Can we use sonication or CTAB to extract the remainder?extract the remainder?
Can we get a reliable band on a Can we get a reliable band on a protein gel?protein gel?
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SonicationSonication
Sonicating water bath – no effectSonicating water bath – no effect Probe sonication (MSE –Soniprep) Probe sonication (MSE –Soniprep)
Strong effect but also caused strong Strong effect but also caused strong local heating effectslocal heating effects
How much heat will CoHo7e-GFP How much heat will CoHo7e-GFP stand?stand?
Is the effect of sonication via heat or Is the effect of sonication via heat or ultrasonics?ultrasonics?
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Heat Resistance of Core-GreenHeat Resistance of Core-Green
0
100000
200000
300000
400000
500000
600000
20C 30C 40C 50C 60C 70C 80C 90C 100C
x64
x128
x256
Green supernatants, pH 8.1 in water, 10 mins at each temperature
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Sonication - heatSonication - heat
10
15
20
25
30
35
40
45
50
0 10 20 30 40 50 60
Time sonicating (sec)
Te
mp
era
ture
(C
)
30ml volume, 10mm probe, start = 16C
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Sonication – extraction of Sonication – extraction of pelletspellets
30ml volume, 10mm probe, start = 16C
0
100000
200000
300000
400000
500000
600000
0 1 2 3 4 5
Time sonicating (min)
Gre
en x8
x16
x32
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SonicationSonication
CoHo7e-GFP is heat stable, 10 mins at 60CCoHo7e-GFP is heat stable, 10 mins at 60C Probe sonication caused strong local Probe sonication caused strong local
heating effects, 50C over 1 minuteheating effects, 50C over 1 minute Sonication caused extraction of green Sonication caused extraction of green
from pellets in < 30 secs (not a heating from pellets in < 30 secs (not a heating effect)effect)
Sonication had no effect on green in total Sonication had no effect on green in total preps, decreased green in pellets and preps, decreased green in pellets and increased green in soluble fractionsincreased green in soluble fractions
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CTABCTAB
Previous work had showed CTAB to Previous work had showed CTAB to help solublize green at high and low help solublize green at high and low concentration but not at 1mMconcentration but not at 1mM
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CTAB – extraction of GreenCTAB – extraction of Green
Water extraction, in 25mM HEPES pH 8
0
20
40
60
80
100
120
% s
olu
ble
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CTAB effect on Protein GelsCTAB effect on Protein Gels
CTAB appeared to destroy larger protein bands
CTAB0.4 4mMpH 6 pH 8 Sonic
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Protein GelsProtein Gels
Can we detect Core-GFP on a protein Can we detect Core-GFP on a protein gel?gel?
Tested effect of protein concentration Tested effect of protein concentration (serial dilution)(serial dilution)
Tested effect of prep temperatureTested effect of prep temperature Tested effect of non-reducing/ reducingTested effect of non-reducing/ reducing Best results – reducing gels 95C for 10 Best results – reducing gels 95C for 10
minsmins
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X33 and E1 soluble fractions
X33 Control Core-GFP E1
66kDa
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Core and Core-HACore and Core-HA Core = 37kDa, Core-HA = 67kDaCore = 37kDa, Core-HA = 67kDa No comparable bands seen on gelsNo comparable bands seen on gels Tried 100C SDS treatment in Tried 100C SDS treatment in
reducing gelsreducing gels Tried high pH, low ionic strengthTried high pH, low ionic strength Tried CTABTried CTAB Tried sonicationTried sonication
Are these proteins not formed, or not soluble?Need an antibody for detection.
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Protein detection - BradfordProtein detection - Bradford
Core – GFP is insoluble at pH 6 but Core – GFP is insoluble at pH 6 but soluble at pH 8.soluble at pH 8.
It appears to be in high concentrationIt appears to be in high concentration Can we detect it using Bradford Can we detect it using Bradford
reagent? reagent? Is there more than in the control?Is there more than in the control? Can we use this to detect Core and Can we use this to detect Core and
Core+HA ?Core+HA ?
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Detection of Core-GFP using Detection of Core-GFP using BradfordBradford
Water extraction pH 8.0
0
0.2
0.4
0.6
0.8
1
1.2
Control Core GFP F1 - HA Eden - HA
Pro
tein
(OD
595
nm)
Pre-sonicated
Sonicated
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Antibody Method - Detection of Antibody Method - Detection of Core ProteinCore Protein
Antibody to Core protein from AbCamAntibody to Core protein from AbCam Method from AadilMethod from Aadil Caution as to non-specific binding of Caution as to non-specific binding of
yeast proteinsyeast proteins Dot blot method established, based on Dot blot method established, based on
Aadil’s method, calibrated used Core-Aadil’s method, calibrated used Core-HA protein from BarryHA protein from Barry
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Antibody detection of core Antibody detection of core proteinprotein
Control Control Eden – HA E1 - GFP Total Soluble Tot Sol Tot Sol Tot Sol
X1
X5
X25
X125
x625
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ConclusionConclusion
We can detect Core protein from E1, We can detect Core protein from E1, CoHo7e-GFP, and from Eden - CoHo7e-CoHo7e-GFP, and from Eden - CoHo7e-HAHA
Amounts detected ~ 1200mg/l E1, Amounts detected ~ 1200mg/l E1, 200mg/l Eden – HA200mg/l Eden – HA
Large proportion of both in the soluble Large proportion of both in the soluble fraction at pH 8.0fraction at pH 8.0
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Future WorkFuture Work
Use dot-blot method to screen all Use dot-blot method to screen all transformants for production of core transformants for production of core (i.e. tandem Core, Core, Core + GFP, (i.e. tandem Core, Core, Core + GFP, Core + HA.Core + HA.
Where no protein found in Where no protein found in transformants, check vector construct transformants, check vector construct and yeast insert by DNA sequencingand yeast insert by DNA sequencing
Optimise production of Core-HA, as Optimise production of Core-HA, as detected by dot-blot detected by dot-blot