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

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

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

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

CoHo7e-GFP in P. pastoris, 5 days induction

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

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

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

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

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?

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?

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

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

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

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

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

CTAB – extraction of GreenCTAB – extraction of Green

Water extraction, in 25mM HEPES pH 8

0

20

40

60

80

100

120

% s

olu

ble

CTAB effect on Protein GelsCTAB effect on Protein Gels

CTAB appeared to destroy larger protein bands

CTAB0.4 4mMpH 6 pH 8 Sonic

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

X33 and E1 soluble fractions

X33 Control Core-GFP E1

66kDa

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.

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 ?

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

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

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

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

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