tsb q7 meeting 02-jun-2009 hepatacore iqur leeds progress
TRANSCRIPT
Overview
• Tandem core production» HA-tandem trials and tribulations
• Dual tandem production» eGFP-tandem» sAg,eGFP-dual tandem
• Space(r) Tandem core» HAVP1-tandem core» sAg-tandem core
• Future work
Bacteria expression
• Sizes of the tandems discussed:
• CoHo7e, HA1s (67kDa)• CoHo7e, eGFP (64kDa)• CoHo7sAg, eGFP (69kDa)• CoHo7e, sAg (spacers) (~43kDa)• CoHo7e, HAVP1 (spacers) (~128kDa)
Batch 7 HA – Tandem core purificationDiscontinuous sucrose gradient
Load
Pel
let
2 3 4 5 6 7 8 9 10 11 12 30M
Bottom Top
1ml fractions40%60% 30%
Load
Pel
let
2 3 4 5 7 8 9 10 11 126
Bio
tin M
r
Cores band at the 60:40% interface
Making HA-tandem… or not
• Adapted from the novel coHo7sAg purification method (see Q6 presentation), a similar high pH – low pH approach was attempted with HA-tandems
• Also attempted higher pH, lower buffer concentration
• Removal of incompatible reagents (Tween 20 precipitates with AmSO4)
• Increasing induction temperature to decrease heavy sediments (16 raised to 27oC)
An example of how NOT to do it…
2 3 4 5 6 7 8 9 10 11 31PM
Bottom Top
1ml fractions40%60% 30%
Eden cell pellet16oC inductionHigh pH lysis (pH8.5)=> Heavy sediment
2 3 4 5 6 7 8 9 10 11PLoadM
Bottom Middle
1ml fractions40%60%
iQur cell pellet27oC inductionNormal pH lysis (pH7.5)=> Lower overall yield
67kDa
HA-Tandem (+ fiends) shipped
HA-tandem
50
37
150
250
25
75
20
100
15
10
50
37
150
250
25
75
20
100
15
10
50
37
150
250
25
75
20
100
15
10
S/N PM
Lessons learnt for HA-tandem
• High pH adversely affects HA1 tandems core sedimentation
• Altered morphology can not be recovered by lowering pH
• Tween 20 is recommended at the start (post lysis) to prevent aggregates
• Low temperature expression levels yield better final product
» (less co-purifying ~37kDa contaminant, which is probably further reduced by differential precipitation with Ammonium Sulphate)
Green Dual tandem - Intro
• Dual construct needed to show “USP”
• eGFP correctly folded fluorescent activity present
• Easy to detect
• Both HBsAg and eGFP inserts have previously been shown to elicit immune responses
• Can be used for preliminary studies in non human primates
Dual tandem cloning
• pET28b-coHe7sAg,eGFP
Core II (aa1-185)
pET 28b-CoHe7e
HiseGFP
Core I (aa1-149)
Nco I Bam HI Not I Eco RI Xho ISac I Sal I
Flexible linker
Nhe
Core II (aa1-185)
pET 28b-CoHe7e,eGFP
His
Hetero tandem
Nhe I
pET 28b-CoHo7sAg,e
HissAg
Nco I Bam HI Not I Eco RI Xho ISac ISal I
Flexible linker
pET 28b-
His
Digest EcoRI/NheI
Homo tandem
Expression not optimal at 37 ˚C
M MNI
M M
I NI I
NI I NI I NI I
NI I
75kDa
25kDa
pET coHo7e
pET coHo7e,
eGFP
pET coHo7sAg,
eGFP pET coHo7e
pET coHo7e,
eGFP
pET coHo7sAg
,eGFP
75kDa
25kDa
75kDa
25kDa
Small scale expression
• Growth to an OD of 0.7 at 37˚C
• Induction with 1mM IPTG for 4h at 37˚C
60
40
30
cores
Analyses:BradfordSDS-PAGEWestern blotELISATEM
IPTG
French Press
14000 psi
Crude Dual tandem core prep
16oC
Sonicat
ion
Lysis in Tris pH8, 5% glycerol, 5mM DTT, Prot. Inhib, benzonase:
Clarification: 26k x g spin
Soluble
Insoluble
2 passesQuick
Pellet30%sucrose
+ 150mM NaCl
pET coHo7e,eGFP
pET coHo7sAg,eGFP
M NI I M NI I
M: Marker; NI: Non Induced; I: Induced
Expression
M T T M I S I S
250 150 100
75
50
37
25 20
10
15
pET coHo7e,eGFP
pET coHo7sAg, eGFP
M: Marker; T: Total Lysate; I: Insoluble Lysate; S: Soluble Lysate
Solubility
Most of the protein is soluble
Large scale prep (at 16oC)
250 150 100
75
50
250 150 100
75
50
M : Marker; L: Load; S/N: Supernatant; P : Pellet
M L L M S/N P S/N P
pETcoHo7e, eGFP
pETcoHo7sAg, eGFP
250
100 75
50
150
37
25 20
10
15
L M S/N P S/N P
250
100 75
50
150
37
25 20
10
15
M L
pETcoHo7e, eGFP
pETcoHo7sAg, eGFP
Western blot: 10E11 dil.1/4000; Goat anti mouse dil.1/2000M: Marker; L: Load; S/N: Supernatant; P: Pellet
Particulate material inferred by sedimentation
pETcoHo7e,eGFP and pETcoHo7sAg,eGFP Bradford assay
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 5 10 15 20 25 30 35 40
Fractions
Co
nc
(mg
/mL
)
pETcoHo7e,eGFP
pETcoHo7sAG,eGFP
Discontinuous Sucrose GradientBradford assay
M 7 8 9 10 11 12L P 5 6 13 14
coHo7sAg, eGFP
M 7 8 9 10 11 12L P 5 6 13 14
coHo7e, eGFP
Discontinuous Sucrose GradientSDS-PAGE Coomassie stained gels
Broad range of sedimentation for both tandems
M 2 5 7 9 11 12 14 18 22 26 30 34
60% 40% 30%1ml fractions
Bottom TopBottom
coHo7sAg, eGFP (69kDa)
250150
10075
5037
2520
15
M 2 5 7 9 11 13 14 18 22 26 3430
1ml fractions60% 40% 30%
TopBottom
coHo7e, eGFP (64kDa)
250150
10075
50
37
2520
Antigenicity of gradient fractions e, eGFP (1)
pETcoHo7e, eGFP Disco. Gradient ELISA
0
0.05
0.1
0.15
0.2
0.25
0 5 10 15 20 25 30 35 40
Fractions
Ab
sorb
ance
Anti sAg
0
0.2
0.4
0.6
0.8
1
1.2
0 5 10 15 20 25 30 35 40
Fractions
Ab
sorb
ance
Anti eGFP
Anti core
Antigenicity of gradient fractions sAg,eGFP (2)
pETcoHo7sAg,eGFP Disco. Gradient ELISA
0
0.05
0.1
0.15
0.2
0.25
0 5 10 15 20 25 30 35 40
Fractions
Ab
sorb
ance
Anti sAg
0
0.2
0.4
0.6
0.8
1
1.2
0 5 10 15 20 25 30 35 40
Fractions
Ab
sorb
ance
Anti eGFP
Anti core
Preparation of pooled fractions
e, eGFP sAg, eGFP
50
37
150
250
25
75
20
100
15
10
S/N PM
50
37
150
250
25
75
20
100
15
10
S/N PM
Sucrose cushion pellet represents final preparation
Formulated in Tris (20mM, pH8) buffer with salt (150mM) and 40% sucrose
Major co-purifying contaminant at ~37kDa
sAg and HAVP1 space(r) types
Core I : atctggaaggatccgccggcggcggccgcgatccgg
L E G S A G G G R D P A
BamHI
Core II: gggtcggcaataatctggaattcgccggcgctagcgacEcoRI NheI
N L E F A G A S D P A S
NotI
Antigen insert site II
Nhe IEco RI
HAVP1
Afl II
TYPE AAntigen insert site II
Nhe IEco RI
HAVP1
KpnI
TYPE C
Antigen insert site II
Nhe IEco RI
HAVP1
KpnIAfl II
TYPE B
Antigen insert site II
Nhe IEco RI
sAg
Afl II
Antigen insert site II
Nhe IEco RI
sAg
KpnI
Antigen insert site II
Nhe IEco RI
sAg
KpnIAfl II
16C
Uni
nd T
ype
A
30C
O
/N T
ype
A
16C
Uni
nd T
ype
C
30C
Uni
nd T
ype
A
30C
Uni
nd T
ype
B
30C
Uni
nd T
ype
C
30C
O/n
Typ
e C
Mar
ker
16C
Uni
nd T
ype
B
16c
O/N
Typ
e B
16C
O/N
Typ
e C
16C
O/N
Typ
e A
30C
O/N
Typ
e B
25015010075
50
37
2520
128 kDa
Blot of HAVP1 Spacers expression at 16°C and 30°C
• Type A gives better expression than type B
• Type B produces more lower order species
• Type C least well expressed
• 16oC (weak but visible) better than 30oC (very faint) in all cases
150KDa
100KDa
16C
Tot
al
Mar
ker
Mar
ker
16C
Sol
uble
16C
Ins
olub
le
16C
Tot
al
16C
Tot
al
16C
Sol
uble
16C
Ins
olub
le
16C
Sol
uble
16C
Ins
olub
le
Type A Type B Type C
* * *
* Sample not correctly normalised (15-fold conc)
HAVP1 space(r) solubility shows little variation
• Solubility [more > less] follows [C > A > B]• Types A and C appear slightly more soluble than type B• Type A more abundant than the others
• Difficult to assess due to concentrated insoluble fraction and over exposure of blot
sAg spacer expression
M U 4 h
rs
O/N UU 4 h
rs
O/N
O/N
4 h
rs
M
50 KDa
37 KDa
Expression at 16 degree Celsius
42 KDa
Type A Type B Type C
Elute immobilised
fraction
VLPs
Immunoaffinity purification
• Possible generic purification approach capturing with anti-core
• Select for correctly folded inserts using conformation-dependent antibodies
• Typically low yields • Very expensive• Dependent on good
reagents
Wash off non-specific
contaminants
HCP
Load sampleImmobilise antibody