Homotandem Core-HA (and Homotandem Core-HA (and Core-GFP) Expression in YeastCore-GFP) Expression in Yeast

Malcolm Stratford & Hazel SteelsMalcolm Stratford & Hazel Steels

MOLOGICMOLOGIC

ContentsContents

BackgroundBackground pH/salt effects on solubilitypH/salt effects on solubility Extraction methods for Core-HAExtraction methods for Core-HA Agitation effects on Core-HAAgitation effects on Core-HA Antioxidants and protease inhibitorsAntioxidants and protease inhibitors Yeast Nitrogen Base media effects Yeast Nitrogen Base media effects

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

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

Salt/pH effect on Core-GFP Salt/pH effect on Core-GFP solubilitysolubility

Starting with a large quantity of soluble Core-GFP, pH 8 in water.

Added to matrix of pH 6.0-8.0, NaCl 0 – 160mM, mixed for 1.5 hours.

Green fluorescence measured in total,soluble and insoluble fractions.

Salt/pH matrix effect on re-Salt/pH matrix effect on re-precipitating Core-GFPprecipitating Core-GFP

20 40 60 80 100 120 140 1606

6.5

7

7.5

8

Ionic strength (mM)

pH60-80

40-60

20-40

0-20

% insoluble

Salt/pH effect on Solubility of Salt/pH effect on Solubility of Core-GFPCore-GFP

Dissolving Core-GFP required high pH and low ionic strength.

This effect is fully reversible, by lowering pH and raising ionic strength

It looks as if VLPs are a half-way stagebetween solubility and insolubility

Induction and extractionInduction and extractionof Core-HAof Core-HA

Time Course E1 Core-GFPTime Course E1 Core-GFP

0

100000

200000

300000

400000

500000

600000

0 3 5 7 10 12 14

Time (Days

Gre

enx64

x128

x256

0 1 2 3 5 7 9 11 days

Time Course 655 = Core-HATime Course 655 = Core-HA

0 1 2 3 5 7 9 11 days

Control 654

655 strain

Time Course 655 = Core-HATime Course 655 = Core-HA

0 1 2 3 5 7 9 11 days

x1

X5

X25

x125

Time Course 655 Core-HATime Course 655 Core-HA

0

50

100

150

200

250

0 2 4 6 8 10 12

Time (Days)

Dilu

tions

Core-HA - Extraction MethodsCore-HA - Extraction Methods

Freeze/Thaw does this damage Core-Freeze/Thaw does this damage Core-HA?HA?

Dissolving out Core-HA – does this need Dissolving out Core-HA – does this need more work?more work?

Will sonication help?Will sonication help?

Time Course of InductionTime Course of Inductionof Core-HA at different of Core-HA at different

agitation ratesagitation rates

Normally faster agitation results in faster protein production, but this will cause increased oxidative stress

GFP induction in P.pastoris; GFP induction in P.pastoris; effect of aerationeffect of aeration

0 2 4 6 10

60100

140180

0

100000

200000

300000

400000

500000

600000

Gre

enn

ess

Time (days)

RPM

60

100

140

180

Core-HA affected by AgitationCore-HA affected by Agitation

0

40

80

120

160

200

0 2 4 6 8

Time (days)

Dilu

tions

80

100

140

180

RPM

AntioxidantsAntioxidants

BHA – Butylated Hydroxy AnisoleBHA – Butylated Hydroxy Anisole Ascorbic acidAscorbic acid EDTAEDTA No discernable effect on protein No discernable effect on protein

production (BHA is toxic)production (BHA is toxic)

Protease InhibitorsProtease Inhibitors

Protease Inhibitor General (Sigma)Protease Inhibitor General (Sigma) Protease Inhibitor Fungal (Sigma)Protease Inhibitor Fungal (Sigma) Z-Leu-Leu-norvalinalZ-Leu-Leu-norvalinal Calpain Inhibitor 1Calpain Inhibitor 1 No discernable effect on protein No discernable effect on protein

production (Fungal Protease is toxic)production (Fungal Protease is toxic)

Yeast Nitrogen BaseYeast Nitrogen Base

S a l t s N i t ro g e n s o u rc e sP o t a s s iu m p h o s p h a t e K H 2 P O 4 1 g A m m o n iu m s u lp h a t e 5 gM a g n e s iu m s u lp h a t e 0 . 5 g H is t id in e H C l 1 0 m gS o d iu m c h lo r id e 0 . 1 g M e t h io n in e 2 0 m gC a lc iu m c h lo r id e 0 . 1 g T ry p t o p h a n 2 0 m g

T ra c e e le m e n t s V i t a m in sB o r ic a c id 5 0 0 u g B io t in 2 u gC o p p e r s u lp h a t e 4 0 u g C a lc iu m p a n t o t h e n a t e 4 0 0 u gP o t a s s iu m io d id e 1 0 0 u g F o l ic a c id 2 u gF e rr ic c h lo r id e 2 0 0 u g In o s i t o l 2 m gM a n g a n e s e s u lp h a t e 4 0 0 u g N ia c in 4 0 0 u gS o d iu m m o ly b d a t e 2 0 0 u g p -A m in o B e n z o ic a c id 2 0 0 u gZ in c s u lp h a t e 4 0 0 u g P y r id o x in e H C l 4 0 0 u g

R ib o fla vin 2 0 0 u gT h ia m in e H C l 4 0 0 u g

Core-HA affected by AgitationCore-HA affected by Agitation

0

40

80

120

160

200

0 2 4 6 8

Time (days)

Dilu

tions

80

100

140

180

RPM

Yeast Nitrogen Base, salts Yeast Nitrogen Base, salts single excess additions, Day 7single excess additions, Day 7

Dilution

- C on t ro l 10P o tas s ium phos pha te K H 2P O 4 50m g 1 .5g 2M agnes ium s u lpha te 50m g 1 .5g 10S od ium c h lo ride 50m g 1 .5g 10C a lc ium c h lo ride 50m g 1 .5g 10S orb ito l 0 .5g 15g 5P o tas s ium c h lo ride 50m g 1 .5g 10G ly c e ro l 100m g 3g 10P o tas s ium iod ide 1m g 30m g 10Zinc s u lpha te 1m g 30m g 10B oric ac id 1m g 30m g 15F erric c h lo ride 1m g 30m g 7M anganes e s u lpha te 1m g 30m g 10S od ium m o ly bda te 1m g 30m g 10C opper s u lpha te 1m g 30m g 10

Dilution

Nitrogen Sources, single excess Nitrogen Sources, single excess additions, Day 7additions, Day 7

Compound per flask per litre Dilution to 0 - Control 10Ammonium sulphate 0.15g 4.5g 10Glycine 10mg 0.3g 30Cysteine 10mg 0.3g 1Leucine 10mg 0.3g 20Methionine 10mg 0.3g 10Histidine 10mg 0.3g 10Asparagine 10mg 0.3g 10Proline 10mg 0.3g 15Adenine 10mg 0.3g 10Aspartic acid 10mg 0.3g 10Arginine 10mg 0.3g 15Tryptophan 10mg 0.3g 10Lysine 10mg 0.3g 10Glutamine 10mg 0.3g 10Phenylalanine 10mg 0.3g 10Isoleucine 10mg 0.3g 10Serine 10mg 0.3g 10Alanine 10mg 0.3g 10Threonine 10mg 0.3g 10

Vitamins, single excess Vitamins, single excess additions, Day 7additions, Day 7

C o m p o u n d p e r f l a s k p e r l i t r e D i l u t i o n t o 0 - C o n t r o l 1 0p - A m i n o B e n z o i c a c i d 1 m g 3 0 m g 1 0B i o t i n 1 m g 3 0 m g 1 0N i a c i n 1 m g 3 0 m g 2 0T h i a m i n e H C L 1 m g 3 0 m g 1 0P y r i d o x i n e H C l 1 m g 3 0 m g 1 0P a n t o t h e n i c a c i d 1 m g 3 0 m g 1 0F o l i c a c i d 1 m g 3 0 m g 1 0In o s i t o l 1 m g 3 0 m g 1 0U r a c i l 1 0 m g 0 . 3 g 1 0

Mixtured nutrients excess Mixtured nutrients excess additions, Day 7additions, Day 7

C o m p o u n d p e r f l a s k p e r l i t r e D i l u t i o n t o 0 - C o n t r o l 1 0B a c t P e p t o n e 5 0 m g 1 . 5 g 1 0T r y p t o n e 5 0 m g 1 . 5 g 1 0C a s e i n 5 0 m g 1 . 5 g 5Y e a s t e x t r a c t 5 0 m g 1 . 5 g 4 0M a l t e x t r a c t 5 0 m g 1 . 5 g 5Y e a s t N i t r o g e n B a s e 0 . 3 g 9 g 5 0V i t - f r e e Y e a s t N B a s e 0 . 3 g 9 g 5B a s e , n o A m m o n i u m o r A A 0 . 0 7 6 g 2 . 2 g 1 0

Conclusions so farConclusions so far

Nitrogen sources such as glycine seem Nitrogen sources such as glycine seem importantimportant

Vitamins seem important, but little effect Vitamins seem important, but little effect when added individually – multi-vitamin when added individually – multi-vitamin effect?effect?

Yeast Extract has a positive effect – Yeast Extract has a positive effect – vitamins?vitamins?

Phosphates are antagonisticPhosphates are antagonistic

Optimised ConcentrationsOptimised Concentrations

Glycine - 0.3g/lGlycine - 0.3g/l Vitamin mixture (x1000) – 100ul Vitamin mixture (x1000) – 100ul

(30ul normal)(30ul normal) Yeast Extract – 4.5g/lYeast Extract – 4.5g/l Yeast Nitrogen Base – 7g/lYeast Nitrogen Base – 7g/l

(6.7g/l normal)(6.7g/l normal)

Time Courses – improved Time Courses – improved induction mediainduction media

0

100

200

300

400

500

600

700

800

0 1 2 3 4 5

Time (days)

Dilu

tions

Glycine

Vitamins

Both

Future WorkFuture Work

Optimize Core-HAOptimize Core-HA Which vitamins? Drop-out experimentsWhich vitamins? Drop-out experiments Combination matrix vitamins/glycineCombination matrix vitamins/glycine Temperature effect, pH effectsTemperature effect, pH effects Run Core-HA on gelsRun Core-HA on gels Purify, look for VLPsPurify, look for VLPs