aex & purification. purification update a reproducible method for vlp purification has been...
TRANSCRIPT
AEX & Purification
Purification update
• A reproducible method for VLP purification has been developed which routinely produces material of 80% plus purity
• However, VLP morphology is not homogenous suggesting some large core-containing complexes are formed
• It is possible that these complexes only form transiently so the time of harvest may need optimisation
• A final “clean-up” step may be required
• Sucrose gradients have been used to generate material for in vivo studies but these are not GMP scaleable
• A combination of SEC and AEX would be ideal but has proven problematic
• Finally, we need to quantify both purity and morphology on a routine basis and so a matrix of data has been proposed
Optimal harvest time
KM71H pHe7 HA2.3,(M2e)3C17-19S.SP UCL fermenter Runs assembly test
• Samples from UCL of fermenter runs 16 (K1,K1 NB1 Harvest sample only) and 20 (HA2.3,(M2e)3C17-19S.SP)
• Samples 1 pre-induction sample.3 samples taken over the induction period at
various times.1 Harvested cell paste sample.
• 2ml each sample run on CL4B XK16/20 in 20mM Tris Ph8.4, 5mM EDTA, 1M Urea
UV 1_280_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e... UV 2_254_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e... UV 3_350_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e...Fraction_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e... Injection_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2... Run Log_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e)...
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UV 1_280_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e... UV 2_254_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e... UV 3_350_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e...Fraction_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e... Injection_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2... Run Log_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e)...
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KM71H pHe7 HA2.3,(M2e)3C17-19S.SP UCL fermenter Runs assembly test CL4B.
UV 1_280_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e... UV 2_254_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e... UV 3_350_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e...Fraction_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e... Injection_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2... Run Log_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e)...
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UV 1_280_Chrom.1:21-11-14 KM71H phe7 HA2.3,(M2e... UV 2_254_Chrom.1:21-11-14 KM71H phe7 HA2.3,(M2e... UV 3_350_Chrom.1:21-11-14 KM71H phe7 HA2.3,(M2e...Fraction_Chrom.1:21-11-14 KM71H phe7 HA2.3,(M2e... Injection_Chrom.1:21-11-14 KM71H phe7 HA2.3,(M2... Run Log_Chrom.1:21-11-14 KM71H phe7 HA2.3,(M2e)...
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UV 1_280_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e... UV 2_254_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e... UV 3_350_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e...Fraction_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e... Injection_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2... Run Log_Chrom.1:21-11-14 KM71H pHe7 HA2.3,(M2e)...
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UV 1_280_Chrom.1:21-11-14 KM71H pHe7 K1,K1 16 H... UV 2_254_Chrom.1:21-11-14 KM71H pHe7 K1,K1 16 H... UV 3_350_Chrom.1:21-11-14 KM71H pHe7 K1,K1 16 H...Fraction_Chrom.1:21-11-14 KM71H pHe7 K1,K1 16 H... Injection_Chrom.1:21-11-14 KM71H pHe7 K1,K1 16 ... Run Log_Chrom.1:21-11-14 KM71H pHe7 K1,K1 16 H ...
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73.5hrs Harvest 75.25hrs
K1,K1 Harvest
Time 0 4.58hrs 26.9hrs
KM71H pHe7 HA2.3,(M2e)3C17-19S.SP UCL fermenter Runs assembly test CL4B. Anti-core blot
rHBc
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#4 #6 #7 #8 #9 #10
#11
#5 #12
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#5 #12
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Time 0 4.58hrs 26.9hrs
73.5hrs K1,K1 Harvest
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Harvest 75.25hrs1 min exposure
1 min exposure
1 min exposure 1 min exposure
1 sec exposure1 min exposure
VLP VLP VLP
VLP VLP VLP
KM71H pHe7 HA2.3,(M2e)3C17-19S.SP UCL fermenter Runs assembly test CL4B. Silver stain of peak Void volume fraction (#4) from each run
• These data suggest that optimal expression is found around 24hrs post-induction
• This agrees with shake flask fermentation
• Expression decreases after this time
Vaccine candidate yeast can be grown in fermenters
Sucrose gradients
Purification of VLPs from CL4B-S1000 columns by sucrose gradientVLP samples (all from standard CL4B – S1000 column preps)• KM71H pHe7 K1,K1, 1.4mg/ml.• BL21 coHe7 HA2.3,e, 0.8mg/ml.• BL21 coHe7 HA2.9,e, 0.7mg/ml.
0.5ml each loaded onto 10 – 60% sucrose gradients (13ml) in 20mM Tris pH8.4, 5mM EDTA.
Centrifugation conditions:Beckman SW40 Ti rotor30,000 rpm 3hrs 4oC
RCF (avg) 113652RCF (max)160070k-Factor 244.1
0.5ml fractions collected from bottom.
Location of core in gradients by dot and western blot
“Goldilocks” VLP region
Summary of results
• The presence of sucrose, particularly in early samples negatively impacted TEM analysis due to low adherence of proteins to the grids and poor staining.
• PHe7K1K1 VLPs increased in numbers from fractions 14 to 18. However, the lower numbers of VLPs in the earlier fractions may have been due to higher concentrations of sucrose (up to 4 %) in these diluted samples. Further dilution is unlikely to improve analysis as the resulting concentration of VLPs would be too low for TEM analysis.
• For the E.coli expressed VLPs, no VLPs were seen in fraction numbers lower than 16. Again, this is most likely due to the high concentration of sucrose in these fractions.
• BL21 CoHe7HA2.3: Large VLP assemblies were seen in fraction 16 with increasing debris present in later fractions.
• BL21 CoHe7HA2.9: VLPs were only seen in fraction 18 but these tended to be within aggregates or clumps.
YEAST produced VLPs passed over sucrose gradient post FPLC: SDS-PAGE/Western Blot of fractions.
Fractions pooled for each construct and sucrose concentration reduced from ~40% to <1% by dilution and re-concentration on 10kDa cut off spin filters.
Fractions of KM71H pHe7 HA2.3,(M2e)3C17.19S.SP also pooled and sucrose reduced to <1% in same way.
Final concentration and volumes.
VLP pool (#): 1.0mg/ml. 1.5ml. Total yield 1.5mgDirty pool (#):2.3 mg/ml. 1.5ml. Total yield 3.4mg
pHe7 HA2.3,(M2e)3C17,19SSucrose gradient purified VLP
Goldilocks VLP region Three bears VLP region
Sucrose cushions can be used to improve the homogeneity of yeast VLP morphology
This process is not perfect but should allow in vivo testing to continue
AEX development-AEX polish of SEC purified VLP
KM71H K1,K1 VLP AEX on CIM-DEAE
UV 1_280_Chrom.1:25-9-14 KM71H pHe7 K1,K1 VLP C... UV 2_254_Chrom.1:25-9-14 KM71H pHe7 K1,K1 VLP C... UV 3_350_Chrom.1:25-9-14 KM71H pHe7 K1,K1 VLP C...Fraction_Chrom.1:25-9-14 KM71H pHe7 K1,K1 VLP C... Injection_Chrom.1:25-9-14 KM71H pHe7 K1,K1 VLP ... Run Log_Chrom.1:25-9-14 KM71H pHe7 K1,K1 VLP CI...
2.32.22.121.91.81.71.61.51.41.31.21.110.90.80.70.60.50.40.30.20.10
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Input run directly through UV detector before loading onto CIM-DEAE
All protein bound but nothing eluted by salt gradient or 1M NaOH
UV 1_280_Chrom.1:25-9-14 KM71H pHe7 K1,K1 VLP p...UV 2_254_Chrom.1:25-9-14 KM71H pHe7 K1,K1 VLP p...Cond_Chrom.1:25-9-14 KM71H pHe7 K1,K1 VLP pH7.0...Conc B_Chrom.1:25-9-14 KM71H pHe7 K1,K1 VLP pH7...PreC pressure_Chrom.1:25-9-14 KM71H pHe7 K1,K1 ... Fraction_Chrom.1:25-9-14 KM71H pHe7 K1,K1 VLP p...Injection_Chrom.1:25-9-14 KM71H pHe7 K1,K1 VLP ... Run Log_Chrom.1:25-9-14 KM71H pHe7 K1,K1 VLP pH...
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• Using prep from high copy clone.• VLP positive fractions from S1000 (41-50)
concentrated to 0.58mg/ml.• Run on CIMmultus QA-1 monolith column (1ml
column)• Buffer A – 20mM Tris pH 8.4, 5mM EDTA• Buffer B – 20 mM Tris pH 8.4, 5mM EDTA,
1M NaCl• Final wash – 1M NaOH, 1M NaCl• 2ml sample loaded
• Method run (based on column volumes)• Column pre-equilibration (5 CV
Buffer A)• Sample load (2ml)• Column wash (5 CV Buffer A)• Elution (10 CV 0 – 50% linear
gradient Buffer B.• Step to 100% Buffer B (10 CV) • High salt wash (5 CV 100% Buffer
B)• Low salt wash (5 CV Buffer A)• NaOH wash (5 CV 1M NaOH)•
KM71H K1,K1 VLP AEX on CIM-DEAE
UV 1_280_Chrom.1:Manual Run 010 Cond_Chrom.1:Manual Run 010 Conc B_Chrom.1:Manual Run 010 PreC pressure_Chrom.1:Manual Run 010Run Log_Chrom.1:Manual Run 010
11511010510095908580757065605550454035302520151050
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20
10
0
-10
-20ml
mAU
Ma
nu
al R
un
9/2
5/2
01
4 2
:42
:13
PM
+0
1:0
0 R
esu
lt: /D
efa
ultH
om
e/A
KTA
pu
re (
Ma
nu
al)
/Ma
nu
al R
un
0
10
Use
r D
ef...
>
Pa
use
In
fin
ite
min
9/2
5/2
01
4 2
:48
:53
PM
+0
1:0
0 (
Issu
ed
) (M
an
ua
l)>
Pa
use
In
fin
ite
min
9/2
6/2
01
4 9
:26
:52
AM
+0
1:0
0 (
Issu
ed
) (M
an
ua
l)>
Pa
use
In
fin
ite
min
9/2
6/2
01
4 9
:48
:46
AM
+0
1:0
0 (
Issu
ed
) (M
an
ua
l)>
Gra
die
nt 0
.0 {
%B
} 0
.00
{m
in}
(Issu
ed
) (M
an
ua
l)>
En
d 9
/26
/20
14
10
:04
:06
AM
+0
1:0
0 (
Issu
ed
) (M
an
ua
l)>
CIM-DEAE regeneration protocol• Wash 1M NaCl, 1M NaOH (20
CV)• Hold over night in 1M NaCl, 1M
NaOH• Wash 1M NaCl, 1M NaOH (10
CV)
• Wash H2O (20 CV)
• Wash 30% 2-Propanol (20 CV)
• Wash H2O (20 CV)
Hold O/N
Protein is tightly bound to matrix, only partially released by over-night soak in NaOH.Most seems to be bound by hydrophobic interaction since released by 2-Propanol
KM71H HA2.3,(M2e)3C17-19S.SP VLP AEX on CIM-QA
Input run directly through UV detector before loading onto CIM-QA
All protein bound but only 25% of OD280 in input eluted by salt gradient or even after 1M NaOH, 1M NaCl. Therefore, protein lost on column.
UV 1_280_Chrom.1:9-1-15 KM71H pHe7 HA2.3,(M2e)3... Cond_Chrom.1:9-1-15 KM71H pHe7 HA2.3,(M2e)3.C17... Conc B_Chrom.1:9-1-15 KM71H pHe7 HA2.3,(M2e)3.C...PreC pressure_Chrom.1:9-1-15 KM71H pHe7 HA2.3,(... Fraction_Chrom.1:9-1-15 KM71H pHe7 HA2.3,(M2e)3... Injection_Chrom.1:9-1-15 KM71H pHe7 HA2.3,(M2e)...UV 1_280@01,BASEM_Chrom.1:9-1-15 KM71H pHe7 HA2... Run Log_Chrom.1:9-1-15 KM71H pHe7 HA2.3,(M2e)3....
2.32.22.121.91.81.71.61.51.41.31.21.110.90.80.70.60.50.40.30.20.10
700
650
600
550
500
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400
350
300
250
200
150
100
50
0ml
mAU
Waste^
32M
ethod R
un 1/9/2015 2:38:13 P
M +
00:00 M
...
>
End_B
lock (Issued) (P
rocessing) (C
om
pleted)
>Injection valve M
anual load (C
om
pleted)
>
0.54
UV 1_280_Chrom.1:9-1-15 KM71H pHe7 HA2.3,(M2e)3... UV 2_254_Chrom.1:9-1-15 KM71H pHe7 HA2.3,(M2e)3... Cond_Chrom.1:9-1-15 KM71H pHe7 HA2.3,(M2e)3.C17...Conc B_Chrom.1:9-1-15 KM71H pHe7 HA2.3,(M2e)3.C... Fraction_Chrom.1:9-1-15 KM71H pHe7 HA2.3,(M2e)3... Injection_Chrom.1:9-1-15 KM71H pHe7 HA2.3,(M2e)...Run Log_Chrom.1:9-1-15 KM71H pHe7 HA2.3,(M2e)3....
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5
0
-5
-10
-15
-20
-25
ml
mAU
Was
te^̂
25242322212019181716151413
^
11109
^̂
65432>
Con
tinue
1/9
/201
5 2:
47:0
1 P
M +
00:0
0 (I
ssue
d) (
Sys
tem
)>
End
_Blo
ck (
Issu
ed)
(Pro
cess
ing)
(C
ompl
eted
)>
End
_Blo
ck (
Issu
ed)
(Pro
cess
ing)
(C
ompl
eted
)>
End
_Blo
ck (
Issu
ed)
(Pro
cess
ing)
(C
ompl
eted
)>
End
_Blo
ck (
Issu
ed)
(Pro
cess
ing)
(C
ompl
eted
)>
End
_Blo
ck (
Issu
ed)
(Pro
cess
ing)
(C
ompl
eted
)>
End
_Blo
ck (
Issu
ed)
(Pro
cess
ing)
(C
ompl
eted
)>
End
_Blo
ck (
Issu
ed)
(Pro
cess
ing)
(C
ompl
eted
)>
rHBc (
50ng
)
Mar
ker
KM71
H pHe7
HA2.
3,(M
2e) 3.S
PC1719
S
High co
py cl
one
Fracti
on 1
8
Fracti
on 1
7
Fracti
on 1
6
VLP fr
actio
ns d
esalt
ed
1 sec exposure 3 hr exposure
KM71H HA2.3,(M2e)3C17-19S.SP VLP AEX on CIM-QACIM-QA acid wash regeneration protocol• 20mM Tris pH 7.0, 5mM EDTA • 50% Acetic acid• 20mM Tris pH 7.0, 5mM EDTA • 50% Acetic acid• 20mM Tris pH 7.0, 5mM EDTA
UV 1_280_Chrom.1:Method Run 008 UV 2_254_Chrom.1:Method Run 008 Conc B_Chrom.1:Method Run 008 PreC pressure_Chrom.1:Method Run 008Run Log_Chrom.1:Method Run 008
8580757065605550454035302520151050
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40
20
0
-20
-40
-60 ml
mAU
Meth
od R
un 1
/12/2
015 1
1:3
9:4
5 A
M +
00:0
0 M
...
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
Missing protein eluted in 1st acid wash
Although protein levels are low, nucleic acid still appears to be bound to the column
KM71H HA2.3,(M2e)3C17-19S.SP VLP AEX on CIM-QA
CIM-QA Benzonase regeneration protocol• 50mM Tris pH 7.4, 2mM MgCl2,
0.1M NaCl • 200u/ml Benzonase• Hold over-night 37oC (#)• 50mM Tris pH 7.4, 2mM MgCl2,
0.1M NaCl UV 1_280_Chrom.1:Manual Run 010 UV 2_254_Chrom.1:Manual Run 010 Conc B_Chrom.1:Manual Run 010 PreC pressure_Chrom.1:Manual Run 010Injection_Chrom.1:Manual Run 010 Run Log_Chrom.1:Manual Run 010
302826242220181614121086420-2-4-6-8-10-12-14-16-18-20
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0
-5
-10
-15
-20
-25
-30
-35 ml
mAU
Man
ual R
un 1
/12/
2015
6:0
7:24
PM
+00
:00
Re
sult:
/De
faul
tHom
e/A
KTA
pure
(M
anu
al)/
Ma
nua
l Run
010
Use
r D
ef..
.>
Aut
o ze
ro U
V (
Issu
ed)
(Man
ual)
>
Co
lum
n po
sitio
n 2
Do
wn
flo
w (
Issu
ed)
(Man
ual)
>
Sys
tem
flo
w 0
.50
0 {m
l/min
} O
ff (I
ssue
d) (
Ma
nua
l)>
Inje
ctio
n va
lve
Inje
ct (
Issu
ed)
(Man
ual)
>
Inje
ctio
n va
lve
Ma
nua
l loa
d (I
ssu
ed)
(Man
ual)
>
Co
lum
n po
sitio
n B
y-pa
ss D
ow
n fl
ow
(Is
sued
) (M
anua
l)>
Co
lum
n po
sitio
n 2
Up
flo
w (
Issu
ed)
(Man
ual)
>
Inje
ctio
n va
lve
Ma
nua
l loa
d (I
ssu
ed)
(Man
ual)
>
Sys
tem
flo
w 1
.00
0 {m
l/min
} O
ff (I
ssue
d) (
Ma
nua
l)>
End
1/1
3/20
15 1
1:22
:11
AM
+00
:00
(Iss
ued
) (M
anu
al)
>
#
UV 1_280_Chrom.1:13-1-15 CIMmultus QA-1 column ... UV 2_254_Chrom.1:13-1-15 CIMmultus QA-1 column ... Cond_Chrom.1:13-1-15 CIMmultus QA-1 column post...Conc B_Chrom.1:13-1-15 CIMmultus QA-1 column po... PreC pressure_Chrom.1:13-1-15 CIMmultus QA-1 co... Run Log_Chrom.1:13-1-15 CIMmultus QA-1 column p...
10095908580757065605550454035302520151050
4846
4442
40
3836
34323028
2624
2220
18161412
108
6
42
0-2-4-6
-8-10
-12-14-16-18
-20-22
-24-26
-28
-30-32
-34-36
-38-40
-42-44
-46-48-50-52
-54-56
-58 ml
mAU
Met
hod
Run
1/1
3/2
015
11:2
6:13
AM
+0
0:00
Me
thod
: CIM
mu
ltus
QA
-1 c
olu
mn
wa
sh R
esul
t: /D
efau
ltHo
me/
AK
TAp
...>
Ho
ld 1
/13/
201
5 11
:32:
14 A
M +
00:
00 (
Issu
ed)
(M
anu
al)
>
Co
lum
n po
sitio
n B
y-pa
ss D
ow
n fl
ow
(Is
sued
) (M
anua
l)>
End
_Blo
ck (
Issu
ed)
(Pro
cess
ing
) (C
om
ple
ted)
>
Ho
ld 1
/13/
201
5 11
:56:
46 A
M +
00:
00 (
Issu
ed)
(M
anu
al)
>
End
_Blo
ck (
Issu
ed)
(Pro
cess
ing
) (C
om
ple
ted)
>
End
_Blo
ck (
Issu
ed)
(Pro
cess
ing
) (C
om
ple
ted)
>
End
_Blo
ck (
Issu
ed)
(Pro
cess
ing
) (C
om
ple
ted)
>
Da
ta c
olle
ctio
n se
tting
s ad
just
ed fo
r cu
rve
UV
1_
280
.>
End
_Blo
ck (
Issu
ed)
(Pro
cess
ing
) (C
om
ple
ted)
>
CIM-QA acid wash regeneration protocol following Benzonase treatment• 20mM Tris pH 7.0, 5mM EDTA • 50% Acetic acid• 20mM Tris pH 7.0, 5mM EDTA • 50% Acetic acid• 20mM Tris pH 7.0, 5mM EDTA
Post Benzonase treatment acid wash elutes protein (small amount) and further nucleic acid
KM71H HA2.3,(M2e)3C17-19S.SP VLP AEX on CIM-QA at pH 7.0
Input run directly through UV detector before loading onto CIM-QA
Nucleic acid content not significantly reduced by AmS04 ppt
All protein boundNo OD280 in input eluted by salt gradient
VLP precipitated by 40% AmSO4 in attempt to reduce nucleic acid content
UV 1_280_Chrom.1:13-1-15 KM71H pHe7 HA2.3,(M2e)... UV 2_254_Chrom.1:13-1-15 KM71H pHe7 HA2.3,(M2e)... Cond_Chrom.1:13-1-15 KM71H pHe7 HA2.3,(M2e)3 SP...Fraction_Chrom.1:13-1-15 KM71H pHe7 HA2.3,(M2e)... Injection_Chrom.1:13-1-15 KM71H pHe7 HA2.3,(M2e... Run Log_Chrom.1:13-1-15 KM71H pHe7 HA2.3,(M2e)3...
2.32.22.121.91.81.71.61.51.41.31.21.110.90.80.70.60.50.40.30.20.10
62
60
58
56
54
52
50
48
46
44
42
40
38
36
34
32
30
28
26
24
22
20
18
16
14
12
10
8
6
4
2
0
-2
-4 ml
mAUW
aste^
32M
eth
od
R
un
1
/1
3/20
15
2
:4
7:50
P
M +
00
:00
M
eth
od
: Tota
l sa
mp
le m
ea
su
re
men
t via
b
yp
ass R
esu
lt: /D
efa
ultH
o...
>
En
d_B
lock (Issu
ed
) (P
ro
ce
ssin
g) (C
om
ple
te
d)
>In
jectio
n va
lve
M
anu
al lo
ad
(C
om
ple
te
d)
>
UV 1_280_Chrom.1:13-1-15 KM71H pHe7 HA2.3,(M2e)... UV 2_254_Chrom.1:13-1-15 KM71H pHe7 HA2.3,(M2e)... Cond_Chrom.1:13-1-15 KM71H pHe7 HA2.3,(M2e)3.C1...Conc B_Chrom.1:13-1-15 KM71H pHe7 HA2.3,(M2e)3.... PreC pressure_Chrom.1:13-1-15 KM71H pHe7 HA2.3,... Fraction_Chrom.1:13-1-15 KM71H pHe7 HA2.3,(M2e)...Injection_Chrom.1:13-1-15 KM71H pHe7 HA2.3,(M2e... Run Log_Chrom.1:13-1-15 KM71H pHe7 HA2.3,(M2e)3...
56545250484644424038363432302826242220181614121086420
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30
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20
15
10
5
0 ml
mAU
Waste^̂
29
28
27
26
25
24
23
22
21
20
19
18
17
^
15
14
13
^̂
10
9876
^^
432E
nd_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
Data
collection s
ett
ings a
dju
ste
d f
or
curv
e U
V 1
_280.
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End 1
/13/2
015 6
:02:0
0 P
M +
00:0
0 (
Issued)
(Manual)
>
KM71H VLP stability at lower pH
• Intention to use CIM-DEAE to purify yeast VLPs at lower pH. To allow this SEC purified VLPs (stored in 20mM Tris, 5mM EDTA pH 8.4) were buffer exchanged to 20mM Tris pH 7.0 or 50mM Na-Citrate buffer pH 6.0 using CL-4B (XK16/20).
• VLPs are expected to elute in void volume.
• Exchanging K1,K1 to pH 6.0 retains integrity of VLPs as shown by elution in expected region.
But
• Exchanging HA2.3,(M2e)3.C17.19S.SP VLP to pH 6.0 appears to cause VLPs to dissociate to constituent units.
• Exchanging HA2.3,(M2e)3C17.19S.SP VLP to pH 7.0 appears to be causing an intermediate level of instability.
• A secondary observation is that Nucleic acid also moves to the small molecule region of the column. This provides further evidence that the nucleic acid present in the VLP region is there due to it being part off or attached to VLPs.
KM71H VLP stability at lower pH
UV 1_280_Chrom.1:16-1-15 KM71H pHe7 K1,K1 CL4B ... UV 2_254_Chrom.1:16-1-15 KM71H pHe7 K1,K1 CL4B ... Fraction_Chrom.1:16-1-15 KM71H pHe7 K1,K1 CL4B ...Injection_Chrom.1:16-1-15 KM71H pHe7 K1,K1 CL4B... Run Log_Chrom.1:16-1-15 KM71H pHe7 K1,K1 CL4B S...
4644424038363432302826242220181614121086420-2
96
94
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90
88
8684
82
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68
66
64
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58
56
54
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42
40
38
36
34
32
30
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26
24
22
20
18
16
14
12
10
8
6
4
2
0
-2ml
mAU
Waste^
15141312111098765432
Method
Run 1/1
6/2015 2
:28:07 P
M +00:0
0 Metho
d: CL4B
SEC X
K 16-20
Result:
/Default
Home/AK
TApure
(Manu..
.>
End_Blo
ck (Issu
ed) (Pro
cessing
) (Comp
leted)>
End_Blo
ck (Issu
ed) (Pro
cessing
) (Comp
leted)>
End_Blo
ck (Issu
ed) (Pro
cessing
) (Comp
leted)>
End_Blo
ck (Issu
ed) (Pro
cessing
) (Comp
leted)>
UV 1_280_Chrom.1:21-1-15 KM71H pHe7 K1,K1 Stabi... UV 2_254_Chrom.1:21-1-15 KM71H pHe7 K1,K1 Stabi... Fraction_Chrom.1:21-1-15 KM71H pHe7 K1,K1 Stabi...Injection_Chrom.1:21-1-15 KM71H pHe7 K1,K1 Stab... Run Log_Chrom.1:21-1-15 KM71H pHe7 K1,K1 Stabil...
44424038363432302826242220181614121086420
10
9.5
9
8.5
8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
-0.5
-1
-1.5
-2
-2.5
-3
-3.5
-4
-4.5
ml
mAU
Waste^
15141312111098765432
End_Blo
ck (Issu
ed) (Pro
cessing
) (Comp
leted)>
End_Blo
ck (Issu
ed) (Pro
cessing
) (Comp
leted)>
End_Blo
ck (Issu
ed) (Pro
cessing
) (Comp
leted)>
End_Blo
ck (Issu
ed) (Pro
cessing
) (Comp
leted)>
K1,K1 pH 8.4 → 6.0
Fractions from Void peak held at pH 6.0 for 72 hrs and then re- run on CL4BShows that K1,K1 is also unstable at low pH over extended periods
KM71H HA2.3,(M2e)3C17-19S.SP VLP AEX on CIM-DEAE pH 7-4 Step Gradient
KM71H HA2.3,(M2e)3C17-19S.SP VLP AEX on CIM-DEAE pH 7-4 1M NaCl Step Gradient
Core eluted at pH7 1M NaCl is lost on concentration – suggesting aggregation
KM71H HA2.3,(M2e)3C17-19S.SP VLP AEX on CIM-DEAE pH 7-4 1M NaCl Step Gradient
• pH 7 0-1M NaCl gradient • Step pH 4 for 5 CV• Re-ran pH 7 0-1M NaCl gradient
AEX development-AEX on lysate
Fract
ion 3
Fract
ion 4
Fractio
n 18
Fractio
n 19
Fract
ion 1
6
Fract
ion 1
5
rHBc
(50n
g)
Mar
ker
Inpu
t
Fract
ion 6
Fra
ction
17
Fractio
n 20
Note – Only detectable core signal is from fraction at end of sample loading period. Indicating that at this point column had become saturated in ability to bind core. Coomassie stain confirms this for other proteins also.
5 sec exposure 10 min exposure
KM71H HA2.3,(M2e)3C17-19S.SP VLP lysate without urea. AEX on CIM-DEAE 1M NaCl gradient pH 8.4
KM71H HA2.3,(M2e)3C17-19S.SP VLP lysate in 1M urea. AEX on CIM-DEAE 1M NaCl gradient pH 8.4
UV 1_280_Chrom.1:22-1-15 KM71H pHe7 HA2.3,(M2e)... UV 2_254_Chrom.1:22-1-15 KM71H pHe7 HA2.3,(M2e)... Conc B_Chrom.1:22-1-15 KM71H pHe7 HA2.3,(M2e)3....Fraction_Chrom.1:22-1-15 KM71H pHe7 HA2.3,(M2e)... Injection_Chrom.1:22-1-15 KM71H pHe7 HA2.3,(M2e... Run Log_Chrom.1:22-1-15 KM71H pHe7 HA2.3,(M2e)3...
65605550454035302520151050-5
1450
1400
1350
1300
1250
1200
1150
1100
1050
1000
950
900
850
800
750
700
650
600
550
500
450
400
350
300
250
200
150
100
50
0
ml
mAU
Waste^
29
28
27
Waste^
25
24
23
^^
20
19
18
17
16
15
14
^
12
11
10
^̂
765432
Meth
od R
un 1
/22
/201
5 1
1:3
0:2
3 A
M +
00:0
0 M
eth
od: C
IMm
ultus D
EA
E-1
AE
X R
esult: /D
efa
ultH
om
e/A
KT
Apure
(...
>
End
_B
lock (
Issue
d)
(Pro
ce
ssin
g)
(Com
ple
ted
)>
End
_B
lock (
Issue
d)
(Pro
ce
ssin
g)
(Com
ple
ted
)>
End
_B
lock (
Issue
d)
(Pro
ce
ssin
g)
(Com
ple
ted
)>
End
_B
lock (
Issue
d)
(Pro
ce
ssin
g)
(Com
ple
ted
)>
End
_B
lock (
Issue
d)
(Pro
ce
ssin
g)
(Com
ple
ted
)>
End
_B
lock (
Issue
d)
(Pro
ce
ssin
g)
(Com
ple
ted
)>
End
_B
lock (
Issue
d)
(Pro
ce
ssin
g)
(Com
ple
ted
)>
End
_B
lock (
Issue
d)
(Pro
ce
ssin
g)
(Com
ple
ted
)>
End
_B
lock (
Issue
d)
(Pro
ce
ssin
g)
(Com
ple
ted
)>
Fract
ion 3
Fract
ion 4
Fractio
n 19
Fractio
n 20
Fract
ion 1
7
Fract
ion 1
6
rHBc
(50n
g)
Mar
ker
Inpu
t
Fract
ion 6
Fra
ction
18
Fractio
n 23
Note – loaded at half total protein load compared to previous sample.No apparent breakthrough of core, coomassie indicates breakthrough of other proteins.Some core signal detected in gradient at ~40% salt (0.4M NaCl)
10 min exposure
Fracti
on 1
6
Fracti
on 1
7
Fractio
n 17 1
M ur
ea
Fractio
n 18 1
M ur
ea
Fracti
on 2
0
Fracti
on 1
9
rHBc (
50ng
)
Mar
ker
Fracti
on 1
5
Fracti
on 1
8 Fra
ction
16
1M u
rea
Fractio
n 19 1
M ur
ea
10 min exposure
After concentration (4x), core signal was detectable.• Sample
without urea treatment eluting over 55 – 88% salt. Some eluting at 100%.
• Sample with urea treatment eluting at 40%.
KM71H K1,K1 VLP lysate without urea AEX on CIM-DEAE 1M NaCl gradient pH 8.4
UV 1_280_Chrom.1:29-1-15 KM71H pHe7 K1,K1 lysat... UV 2_254_Chrom.1:29-1-15 KM71H pHe7 K1,K1 lysat... Conc B_Chrom.1:29-1-15 KM71H pHe7 K1,K1 lysate ...Fraction_Chrom.1:29-1-15 KM71H pHe7 K1,K1 lysat... Injection_Chrom.1:29-1-15 KM71H pHe7 K1,K1 lysa... Run Log_Chrom.1:29-1-15 KM71H pHe7 K1,K1 lysate...
65605550454035302520151050-5
1850
1800
1750
1700
1650
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1500
1450
1400
1350
1300
1250
1200
1150
1100
1050
1000
950
900
850
800
750
700
650
600
550
500
450
400
350
300
250
200
150
100
50
0
-50ml
mAU
Waste^
29
28
27
Waste^
25
24
23
^^
20
19
18
17
16
15
14
^
12
11
10
^̂
765432
Meth
od R
un 1
/29/2
015 1
0:0
9:5
2 A
M +
00:0
0 M
eth
od:
CIM
multus D
EA
E-1
AE
X R
esult:
/Defa
ultH
om
e/A
KTA
pure
(..
.>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
Fract
ion 3
Fract
ion 4
Fractio
n 18
Fractio
n 20
Fract
ion 1
6
Fract
ion 1
5
rHBc
(50n
g)
Mar
ker
Inpu
t
Fract
ion 6
Fra
ction
17
Fractio
n 23
• Significant core in all of flow-through indicating that column has saturated in ability to bind core (possibly higher core content of K1,K1 or lower affinity for DEAE of K1,K1.)
• Core signal eluting over 20 – 54% salt.
• At higher salt increase in high molecular weight aggregate seen.
1 sec exposure 10 min exposure
KM71H K1,K1 VLP lysate in 1M urea AEX on CIM-DEAE 1M NaCl gradient pH 8.4
UV 1_280_Chrom.1:29-1-15 KM71H pHe7 K1,K1 VLP 1... UV 2_254_Chrom.1:29-1-15 KM71H pHe7 K1,K1 VLP 1... Conc B_Chrom.1:29-1-15 KM71H pHe7 K1,K1 VLP 1M ...Fraction_Chrom.1:29-1-15 KM71H pHe7 K1,K1 VLP 1... Injection_Chrom.1:29-1-15 KM71H pHe7 K1,K1 VLP ... Run Log_Chrom.1:29-1-15 KM71H pHe7 K1,K1 VLP 1M...
65605550454035302520151050-5
800
780
760
740
720
700
680
660
640
620
600
580
560
540
520
500
480
460
440
420
400
380
360
340
320
300
280
260
240
220
200
180
160
140
120
100
80
60
40
20
0
ml
mAU
Waste^
29
28
27
Waste^
25
24
23
^^
20
19
18
17
16
15
14
^
12
11
10
^̂
765432
Meth
od R
un 1
/29/2
015 1
1:1
1:4
3 A
M +
00:0
0 M
eth
od:
CIM
multus D
EA
E-1
AE
X R
esult:
/Defa
ultH
om
e/A
KTA
pure
(..
.>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
Data
colle
ction s
ett
ings a
dju
ste
d f
or
curv
e U
V 1
_280.
>
Data
colle
ction s
ett
ings a
dju
ste
d f
or
curv
e C
ond.
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
End_B
lock (
Issued)
(Pro
cessin
g)
(Com
ple
ted)
>
Data
colle
ction s
ett
ings a
dju
ste
d f
or
curv
e U
V c
ell
path
length
.
Hold
1/2
9/2
015 4
:34:0
1 P
M +
00:0
0 (
Issued)
(Manual)
>
Inle
t A
A2 (
Issued)
(Manual)
>
End 1
/29/2
015 6
:01:0
0 P
M +
00:0
0 (
Issued)
(Manual)
>
Note – by end of this run back-pressure on had increased to a level at which column can no longer be used
Fract
ion 3
Fract
ion 4
Fractio
n 20
Fractio
n 23
Fract
ion 1
7
Fract
ion 1
6
rHBc
(50n
g)
Mar
ker
Inpu
t
Fract
ion 6
Fra
ction
18
Fractio
n 24
1 sec exposure10 min exposure
• Flow through exhibits strange behaviour, core signal decreasing, this cannot happen on AEX unless material is aggregating and fouling column – decrease in signal corresponds to rapid increase in back-pressure.
• Core signal eluting between 40 – 60% salt
KM71H pHe7 K1,K1 High Copy Number Clone lysate and lysate after 1M urea loaded onto CIMmultus DEAE AEX fractions desalted and 4 fold concentrated
Fract
ion 1
6
Fract
ion 1
7
Fractio
n 17 1
M
ureaFrac
tion 1
8 1M
urea
Fract
ion 2
3
Fract
ion 2
0
rHBc
(50n
g)
Mar
ker
Fract
ion 1
5
Fract
ion 1
8 Fra
ction
16
1M u
rea
Fractio
n 20 1
M ur
ea
• K1,K1 binds with lower affinity than HA2.3,(M2e)3C17-19S.SP
• Sample without urea elutes mainly ~ 20 - 50% salt
• With urea elutes over similar salt range.
• Urea does not seem to lower affinity of K1,K1 for DEAE 1 sec exposure 10 min exposure
KM71H HA2.3,(M2e)3C17-19S.SP and K1,K1 VLP Lysate with and without 1M urea AEX on CIM-DEAE
Overall conclusions
• Both constructs bind DEAE
• K1,K1 has lower affinity than HA2.3,(M2e)3C17-19S.SP
• Urea treatment of HA2.3,(M2e)3C17-19S.SP reduces affinity to similar value as K1,K1
• Overall recovery is low for both proteins – large amount of material appears to foul column
• Urea treated K1,K1 loading of column resulted in complete blockage of column – but it is possible that this was result of cumulative fouling rather than specific to K1,K1.
Is urea necessary for SEC preparation?
KM71H pHe7 HA2.3,(M2e)3C17-19S.SP High Copy Clone VLP prep without urea
• Using High Copy clone (as defined by qPCR). Yeast induced without prior starvation period.
• 26g cells resuspended in 320ml lysis buffer (20mM Tris pH 8.4, 2mM AEBSF, 5mM DTT, 3000U Benzonase (93u/g)) lysed by pressure homogenisation 500 bar, 3 passes
• Soluble core extracted from crude lysate with 0.1% Triton X-100 for 1 hr and then centrifuged ~20k x g, 30mins, 4oC
• Supernatant (320ml) diluted with 320ml 20mM Tris pH 8.4, 10mM EDTA, to give a final buffer of 20mM Tris pH 8.4, 5mM EDTA. Note – 1M Urea was omitted from this buffer. Vacuum filtered through 0.8µm, 0.45µm and 0.22µm membrane filters
• Final filtrate washed on 1MDa Pellcon TFF, reducing volume to 25ml. MDa retentate sonicated 2 x 10 sec at 50% power.
• Then filtered through 0.2µm syringe filter followed by 0.1µm syringe filter (filter washes with SDS buffer after use). Significant back pressure observed on passage through 0.2µm and 0.1µm filter. Stored overnight at 4oC. Soincated 2 x 10 sec, Then re-filtered (0.2µm).
• VLPs purified using 2-step Automated AKTAPure program• Filtrate applied to Sepharose 4B column (XK26/92) in 20mM Tris pH 8.4, 5mM EDTA,
Note – no Urea in this stage. • Void volume material (160-205ml corresponding to fractions 4 – 8 in previous method,
45ml) applied to Sephacryl S1000 column (XK50/92) in 20mM Tris pH 8.4, 5mM EDTA,
KM71H pHe7 HA2.3,(M2e)3C17-19S.SP High Copy Clone VLP +/- urea: CL4B comparison to same scale
• Without urea void region is 70% of + Urea, Small protein regions are equal +/- urea• Without urea more large size material is being removed on filters
KM71H pHe7 HA2.3,(M2e)3C17-19S.SP High Copy Clone VLP prep without urea: CL4B Void on XK 50/92 S1000
UV 1_280_Chrom.1:13-2-15 KM71H pHe7 HA2.3,(M2e)... UV 2_254_Chrom.1:13-2-15 KM71H pHe7 HA2.3,(M2e)... UV 3_350_Chrom.1:13-2-15 KM71H pHe7 HA2.3,(M2e)...Fraction_Chrom.1:13-2-15 KM71H pHe7 HA2.3,(M2e)... Injection_Chrom.1:13-2-15 KM71H pHe7 HA2.3,(M2e... Run Log_Chrom.1:13-2-15 KM71H pHe7 HA2.3,(M2e)3...
22002100200019001800170016001500140013001200110010009008007006005004003002001000
100
98
96
94
92
90
88
86
84
82
80
78
76
74
72
70
68
66
64
62
60
58
56
54
52
50
48
46
44
42
40
38
36
34
32
30
28
26
24
22
20
18
16
14
12
10
8
6
4
2
0 ml
mAU
Waste^^
78
^
76
^
74
^
72
^
70
^
68
^
66
^
64
^
62
^
60
^
58
^
56
^
54
^
52
^
50
^
48
^
46
^
44
^
42
^
40
^
38
^
36
^
34
^
32
^
30
^
28
^
26
^
24
^
22
^
20
^
18
^
16
^
14
^
12
^
10
^
8^
6^
Waste^̂̂
Method R
un 2/13/2
015 10:2
7:04 AM
+00:00 M
ethod: S1
000 SEC
XK 50-92
Multi-step
Result: /D
efaultHom
e/A...
>
End_Bloc
k (Issued)
(Process
ing) (Com
pleted)
>
Data colle
ction setti
ngs adjust
ed for cur
ve UV 1_
280.>
End_Bloc
k (Issued)
(Process
ing) (Com
pleted)
>
Data colle
ction setti
ngs adjust
ed for cur
ve UV 1_
280.>
Data colle
ction setti
ngs adjust
ed for cur
ve UV ce
ll path len
gth.
End_Bloc
k (Issued)
(Process
ing) (Com
pleted)
>
Data colle
ction setti
ngs adjust
ed for cur
ve UV 1_
280.>
End_Bloc
k (Issued)
(Process
ing) (Com
pleted)
>
UV 1_280_Chrom.1:20-1-15 KM71H pHe7 HA2.3,(M2e)... UV 2_254_Chrom.1:20-1-15 KM71H pHe7 HA2.3,(M2e)... UV 3_350_Chrom.1:20-1-15 KM71H pHe7 HA2.3,(M2e)...Fraction_Chrom.1:20-1-15 KM71H pHe7 HA2.3,(M2e)... Injection_Chrom.1:20-1-15 KM71H pHe7 HA2.3,(M2e... Run Log_Chrom.1:20-1-15 KM71H pHe7 HA2.3,(M2e)3...
22002100200019001800170016001500140013001200110010009008007006005004003002001000
200
195
190
185
180
175
170
165
160
155
150
145
140
135
130
125
120
115
110
105
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0 ml
mAU
Waste^^
78
^
76
^
74
^
72
^
70
^
68
^
66
^
64
^
62
^
60
^
58
^
56^
54^
52
^
50
^
48
^
46
^
44
^
42
^
40
^
38
^
36
^
34
^
32
^
30
^
28
^
26
^
24
^
22
^
20
^
18
^
16
^
14
^
12
^
10
^
8
^
6
^
Waste^̂̂
Method R
un 1/20/2
015 10:4
0:10 AM
+00:00 M
ethod: S1
000 SEC
XK 50-92
Multi-step
Result: /D
efaultHom
e/AKT...
>
End_Bloc
k (Issued)
(Process
ing) (Com
pleted)
>
Data colle
ction setti
ngs adjust
ed for cur
ve UV 1_
280.>
End_Bloc
k (Issued)
(Process
ing) (Com
pleted)
>
Data colle
ction setti
ngs adjust
ed for cur
ve Cond.
>
Data colle
ction setti
ngs adjust
ed for cur
ve UV ce
ll path len
gth.
End_Bloc
k (Issued)
(Process
ing) (Com
pleted)
>
Data colle
ction setti
ngs adjust
ed for cur
ve Cond.
>
End_Bloc
k (Issued)
(Process
ing) (Com
pleted)
>
0
100
0
200
No Urea
1M Urea
KM71H pHe7 HA2.3,(M2e)3C17-19S.SP High Copy CloneVLP prep without urea CL4B VoidRun on S1000 XK50/92
rHBc
(50n
g)Mar
ker
#70
#38
#44
#47
#50
#53
#56
#59
#41
#65
rHBc
(50n
g)Mar
ker
#2 (I
nput
)#8 #10#18
#20
#23 #26
#29
#32
#35 rH
Bc (5
0ng)
Mar
ker
#70
#38
#44
#47
#50
#53
#56
#59
#41
#65
rHBc
(50n
g)
Mar
ker
#2 (I
nput
)#8 #10#18
#20
#23 #26
#29
#32
#35
No Urea 1M Urea
Increased degradation in absence of urea
Purification of VLPs from CL4B-S1000 columns by sucrose gradient
rHBc
(50n
g)
Mar
ker
#26
#7 #12
#14
#16
#18
#20
#22
#10
#24
rHBc
(50n
g)
Mar
ker
#26
#7 #12
#14
#16
#18
#20
#22
#10
#24
KM71H pHe7 K1,K1
60% 10%
KM71H pHe7 HA2.3,(M2e)3C17.19S.SP
1M Urea Yield = 0.04mg/g
rHBc
(50n
g)
Mar
ker
#26
#7 #12
#14
#16
#18
#20
#22
#10
#24
KM71H pHe7 HA2.3,(M2e)3C17.19S.SP
No Urea Yield = 0.05mg/gWithout Urea more core in ‘Goldilocks’ zone but more degradation – need EMs
Data reporting matrix
KM71H pHe7 HA2.3,M2e Twin High Copy Clone VLP prep
• Using High Copy (~) clone (as defined by qPCR). Yeast induced without prior starvation period.
• 26g cells resuspended in 320ml lysis buffer (20mM Tris pH 8.4, 2mM AEBSF, 5mM DTT, 3000U Benzonase (93u/g)) lysed by pressure homogenisation 500 bar, 3 passes
• Soluble core extracted from crude lysate with 0.1% Triton X-100 for 1 hr and then centrifuged ~20k x g, 30mins, 4oC
• Supernatant (480ml) diluted with 320ml 20mM Tris pH 8.4, 10mM EDTA, 2M Urea to give a final buffer of 20mM Tris pH 8.4, 5mM EDTA 1M Urea. Vacuum filtered through 0.8µm, 0.45µm and 0.22µm membrane filters
• Final filtrate washed on 1MDa Pellicon TFF, reducing volume to 25ml. Unlike other higher copy clones no detectable back pressure detected.
• Then filtered through 0.2µm syringe filter followed by 0.1µm syringe filter (filter washes with SDS buffer after use). Significant back pressure observed on passage through 0.2µm and 0.1µm filter (2 x 0.1µm units used)
• Stored overnight at 4oC. Sonicated 2 x 10 sec, Then re-filtered (0.2µm). • VLPs purified using 2-step Automated AKTAPure program• Filtrate applied to Sepharose 4B column (XK26/92) in 20mM Tris pH 8.4, 5mM EDTA,
1M Urea. • Void volume material (160-205ml corresponding to fractions 4 – 8 in previous method,
45ml) applied to Sephacryl S1000 column (XK50/92) in 20mM Tris pH 8.4, 5mM EDTA,
KM71H pHe7 HA2.3,M2e Twin High Copy Clone VLP prep
20K
s/n
20K
pell
et D
il s/n
0.1
filtra
te
Perm
etat
eM
Da Son
icate
d
CL4B In
put
0.1
filter
SDS w
ash
MDa
0.2
filtra
te
rHBc
(50n
g)
Mar
ker
Crude
lysa
te
1 sec exposure
100 200 300 400 500 600 700 800 900 10000
1
2
3
4
5
6
f(x) = 4.78434844391888 x^-0.500280863942421R² = 0.970418764248172
Flux ml/min
Flux ml/...
Permeate (total vol) ml
Flu
x ra
te (
ml/
min
)
TFF
CL4B
KM71H pHe7 HA2.3,M2e Twin High Copy Clone VLP prep: CL4B Void on XK 50/92 S1000
Fractions pooled and concentrated to 10ml on 1MDa Pellicon. Then to 3ml on 10kDa spin filter.
VLP Pool = #41 - 53, protein concentration 8mg/ml, total yield 24mg
VLP
VLP
Purification of VLPs from CL4B-S1000 columns by sucrose gradient: SDS-PAGE/Western Blot of fractions.
rHBc
(50n
g)
Mar
ker
#26
#7 #12
#14
#16
#18
#20
#22
#10
#24
60% 10%
Fractions pooled and sucrose concentration reduced from ~40% to <1% by dilution and re-concentration on 10kDa cut off spin filters.
Final concentration and volumes.
VLP pool (#): 0.57mg/ml. 1.5ml. Total yield 0.86mgDirty pool (#): 0.35mg/ml. 1.5ml. Total yield 0.53mg
Biochemistry summary data
Parameter Value Units
Volume of culture 1.8 L
Wet weight pellet 26 g
Band visible on initial gel? Y (Y/N)
TFF y = 4.7843x-0.5
R² = 0.9704Equation
AUC (S1000 fractions) 27.5 mAu*ml
Purity (image analysis) 20% %
Amount recovered 24 mg
Yield 0.09 %
Purity sucrose (image analysis) 20% %
Amount recovered sucrose 0.86 mg
Yield sucrose 0.003 %
EM (image analysis) % VLP
Image analysis
S1000 Sucrose
1 648.719 398.284 337.042 2202.77 2119.2052 2115.276 2130.548 2327.861 1584.234 1052.2343 2555.205 2089.598 1987.355 2838.225 1394.447
TC 2494.933 1792.497 1778.355 2164.175 2136.9125 1430.154 2281.962 2519.569 190.263 1617.0546 400.527 133.385 2362.1257 1752.497 2016.125 400.3358 1854.154 851.284 3009.832
Total 13251.47 11693.68 14722.47 8979.667 8319.852% purity 18.8% 15.3% 12.1% 24.1% 25.7% 19.2%
1 1942.598 1846.719 2116.891 2018.0622 3274.912 3911.083 3326.669 3846.79
TC 2152.255 2266.719 1980.184 1596.1844 757.456 899.698 728.163 616.4565 477.335 514.113 382.213 899.1846 1873.912 1464.083 1118.548 259.092
Total 10478.47 10902.42 9652.668 9235.768% Purity 20.5% 20.8% 20.5% 17.3% 19.8%