Homotandem Core-HA (and Core-GFP) Expression in Yeast Malcolm Stratford & Hazel Steels MOLOGIC.
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Transcript of Homotandem Core-HA (and Core-GFP) Expression in Yeast Malcolm Stratford & Hazel Steels MOLOGIC.
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