TSB Q5 Meeting
16-Dec-2008
Hepatacore
iQur Leeds Progress
Overview
• Introduction
• Yeast expression – issues
• Non-adjuvanted coHo7e,HA1s
• Dual tandem core purification attempts
• Alternative ‘flu antigens in tandem core
• Transfer of new promoter; Transfer of dual Hep A/B
• The case for “Spacers”: sAg in Core I versus Core II
• Future work
The tandem core platform
Core I (aa1-149)
Nco I Bam HI Not I Eco RI Xho ISac I Sal I
Flexible linker
Antigen insert site I
Antigen insert site II
Nhe I
Core II (aa1-149)
pET 28b-CoHo7e
His
Homotandem core construct
Monomeric HBcAg (1-149)VLPs
Heterotandem HBcAg VLPs
60nM
Cryo-EM reconstructions of monomeric and tandem core particles. Performed by Dr R. Gilbert
(University of Oxford)
37 KDa
Tandem core proteinFlexible linker
VP
4 VP2 VP3 VP1
HAV P1
Target PathogensHepatitis B virus
• Enveloped virus
• Neutralising antigen surface antigen (HBsAg, aa124-137)
• Current vaccine – yeast expressed HBsAg VLPs
• 5 KDa insert
108
155
• Non-enveloped virus
• Neutralising antigen – cluster of epitopes in VP1 and VP3
• Current vaccines – live attenuated or inactivated whole virus
• 90 KDa insert
Hepatitis A virus
Core I Core IINco I Bam HI Not I Eco RI Nhe I Xho ISac I Sal I
HAV P1 (aa1-791)
Flexible linker
Antigen insert site I
VP
4 VP2 VP3 VP1
125 KDa
Core I Core IINco I Bam HI Not I Eco RI Nhe I Xho ISac I Sal I
HBsAg (108-155)Flexible linker
Antigen insert site I
Antigen insert site II42 KDa
C2PR8HA_F2C2PR8HA_R2
C2PR8HA_F1C2PR8HA_R1
C2PR8HA_F2C2PR8HA_R2
C2PR8HA_F1C2PR8HA_R1
Pathogen and useful model:Influenza Haemagglutinin• H1 serotype (PR8) HA1 globular domain cloned into homo-tandem core• Functional assay to confirm conformation of the haemagglutinin• Protection studies can be done in a mouse model• Express and purify from E.coli for optimisation assays• Optimise haemagglutination, biophysical (EM, CD) and other in vitro
assays
Yeast – PICZ revisited
Yeast expression - Revisited
• Previous experiments may have been overly diluted compared to some values in the literature
• Cross-reactivity observed in previous western blots
• Using pPICZ-C as the expression vector, constructs were transformed and induced and larger pellets were received from Mologic for high cell density lysis by vortex + glass beads
• Non-transformed P. pastoris (X33 strain)• Wt HBc149• CoHo7e,eGFP
• Total and soluble lysates subjected to SDS-PAGE, blotted on to PVDF membrane and probed with several monoclonal antibodies:
• Anti-core: 10E11, (13A9, H5F6 – data not presented)• Anti-EGFP• Anti-mouse secondary (-ve control)
More Yeast Western blotsHigh Cell density Lysis
• Western blot revealed that only eGFP construct was detectable• No evidence of cross reactivity of the secondary antibodies
50
37
75100150250
2520
15 / 10
T S T S T S T S T S T S T S T S T S T S T S T S
X33HBc149 eGFP X33
HBc149 eGFP X33
HBc149 eGFPX33
HBc149 eGFP
Coomassie 10E111/ 10,000 Dil.
Anti-eGFP1/ 4,000 Dil.
2oAb only1/ 5,000 Dil.
IPTG
French Press
15000 psi
Pellet30%sucrose
Method – tandem core prep
27oC
605040302010
cores
20
60
Analyses:BradfordSDS-PAGEWestern blotELISATEM
Sonicat
ion
DRY ICE
MologicLtd.
ARCHIVES 2
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Expression of cores in yeast
A B
WholeLysate
37
50
64
98
22
A
B
Western Blot of samples from lysis and clarification yeast expressed material.
A. CoHe7e B. CoHe7e,eGFP
37
50
64
98
22
A
B
A B
Solublefraction
Sucrosesolublefraction
A B AB B A
Concentrated protein
1. Sand and Liquid nitrogen lysis with benzonase and protease inhibitors
2. Clarify 1: Spun at 26,000 xg
3. Clarify 2: Added sucrose to 0.15M and spun at 150,000 xg
4. Sucrose cushion concentration: supernatant layered over 30% sucrose cushion. Spun at 150,000xg
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It can be done
Non-Adjuvanted Immunogen
• Immunogen = coHo7e,HA1s• Expressed and purified from E.coli
lysates• Material used in immunogenicity
(iQur) and stability studies (Arecor)
• To date immunogen has been mixed with adjuvant = Imject® (aluminium hydroxide, Alum)
• Aggregation and sedimentation hindered previous studies without adjuvant
coHo7e,HA1s sucrose stability
0
0.1
0.2
0.3
0.4
0.5
0.6
Neat S/N Pellet
Sample
Co
nc
(mg
/ml) 0%
10%
20%
40%
• 0%, 10%, 20%, 40% sucrose tandem-HA stored at 2 – 8oC over 8 days
• Samples were centrifuged (12,000 x g) for 10 minutes
• Pellet and supernatant samples were analysed by SDS-PAGE and Bradford
• Addition of sucrose helps resist aggregation and resultant sedimentation of HA-tandem
coHo7e,HA1s minus AdjuvantSucrose stabilisation
M M P S/N P S/N P S/N P S/N
0% 10% 20% 40%
M M P S/N P S/N P S/N P S/N
0% 10% 20% 40%
60
220
100
45
3020
Purification of Dual sAg, HA tandem core
Last time we discussed:• Initial expression and solubility indicated workable amounts• Soluble fraction processed by AmSO4 ppt, sucrose gradients tightly at
40%:60% interface
• Attempted solvent extraction to remove potential lipid contaminants – no effect, meaning protein or CHO
• Plus/ minus detergent (TW20): soluble lysates were analysed for antigenicity (10E11, anti-sAg (mAbs), anti-PR8 (pAb)) demonstrated no preference for detergent
• Core response was strong• sAg weak in comparison to control but titrated• PR8 good, but evidence of strong cross-reactivity E.coli or core
• Strong reducing conditions (beta-mercaptoethanol)
Discontinuous gradient ofcoHo7sAg, HA1s soluble lysate with B-ME
• Strong reducing conditions disrupt tight banding at the 60:40% sucrose interface
M T I S 1 3 5 7 8 9 11 13 15 16 17 19 21 23 24 25 27 29 31 33
Discontinuous sucrose gradient fractions
60
220
100
45
3020
60% 40% 20%Sucrose %
Alternative treatments
• Heat treatment (+/- urea) to clarify away from E.coli proteins
- Urea
T I S HI HSM
+ Urea
T I S HI HSM
72 kDa
Modified Purification I
• coHo7e run alongside sAg,HA dual tandem• Lysed, clarify, heat + clarify, dialysis, sucrose cushion
• Gels, WB, ELISA, Bradford
Lysis and clarify
T I SM I ST
Empty sAg,HA
Heat treatment and clarify
HI HSS HI HSM S
Empty sAg,HA
Modified Purification II
• Start: (Sol lysate) coHo7e = 40 mg coHo7sAg,HA1s = 42.4 mgEnd: (Cushion) coHo7e = 10.8 mg coHo7sAg,HA1s = 4.8 mg (Total Protein)
• Proportion of tandem core in these preps is low at the end of first stage• These results suggests that it is not feasible to purify away from contaminants by
these methods
Dialysed material over sucrose cushion
Cushio
n pe
llet
Cushio
n pe
llet
Empty sAg,HA
AmSO4 ppt
20% 30% 40% 20% 30% 40%
Empty sAg,HA
• Sizes of the tandems discussed:• HBc149 (wild type sequence) (17kDa)• CoHo7e (37kDa)• CoHo7e,eGFP (65kDa)• CoHo7e,HA1s (67kDa)• CoHo7sAg,e (42kDa)• CoHo7sAg,HA1s (72kDa)• CoHo7e,HAVP1 (125 kDa)
• Expression shifted from yeast to bacteria
More Bacteria expression
• Sizes of the tandems discussed:• HBc149 (wild type sequence) (17kDa)• CoHo7e (37kDa)• CoHo7e,eGFP (65kDa)• CoHo7e,HA1s (67kDa)• CoHo7sAg,e (42kDa)• CoHo7sAg,HA1s (72kDa)• CoHo7e,HAVP1 (125 kDa)
• Expression shifted from yeast to bacteria
Expression of HAV P1 (it expressed…!!)
32.5
25
47.5
62
83
175
16.56.5
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E.Coli expression of HAVP1 and sAg
coHo7e
coHo7sAg,e
coHo7e,HAVP1
10E111/ 2,000 Dil.
Anti-HBsAg1/ 1,000 Dil.
50
37
75
100
150
250
252015 / 10
IPTG - + - + - + - + - + - +
Em
pty
HA
VP
1
sAg
Em
pty
HA
VP
1
sAg
- + - + - +
Em
pty
HA
VP
1
sAg
Coomassie
Restriction sites within antigen insertSize issuesFINALLY AFTER MULTIPLE APPROACHES
Dual Hep A/ B candidatecoHo7sAg,HAVP1
Ligations at 1:10 ratio Ligations at 1:20 ratio
PIC
3.5
K c
oH
o7
sA
g,H
AV
P1
HBsAg (108-155)
Nco I Bam HI Not I Eco RI Nhe I Xho ISac I Sal I
HAV P1 (aa 1-791)
VP
4 VP2 VP3 VP1
pET coHo7sAg,HAVP1
Alternative ‘Flu inserts
Neuraminidase (NA) clone verificationViral RNA isolation RT PCR with RT template pJET shuttle Ligate
• Colony screen verified by BamHI digest:• Negative = linearise with BamHI in core I• Positive = Band release cutting BamHI in core I and BamHI internal to NA
sAgEmpty
~1400bp ~1600bp
-ve CTRL
-ve CTRL# 1 - 5 # 1 - 5
• PCR from template containing HA1 long (pJET::HA1l )• Double digest of PCR product (Eco RI – Nhe I) yielding insert• Ligate into coHo7e,HA1s (Eco RI – Nhe I digested)
• Release of HA1s indicates successful digest• Gel purify vector backbone
• Colonies screened by PCR • # 1 - 60
-ve CTRL
HA1-long (HA1l) clone verification
1kb
La
dd
er
10
0b
p L
ad
de
r
Ta
c p
rom
ote
r
PCR
Bgl II – Nco ICore I
(aa1-149)
Nco I Bam HI Not I Eco RI Xho ISac I Sal I
Flexible linker
Antigen insert site I
Antigen insert site II
Nhe I
Core II (aa1-149)
pET 28b-CoHo7e
His
Substitution of T7 promoterin the pET coHo7e vector
Promoter ‘Flu Hep A/B
tac-coHo7e coHo7e,NA coHo7sAg,HAV
tac-coHo7sAg,e coHo7e,M2e coHo7HAV,e
All the others… coHo7e,HA1l
Promoter ‘Flu Hep A/B
tac-coHo7e coHo7e,NA coHo7sAg,HAV
tac-coHo7sAg,e coHo7e,M2e coHo7HAV,e
All the others… coHo7e,HA1l
Cloning summary
sAg – Core I vs Core IISecond look
• Core I has extra residues around the e1 loop due to Not I restriction site being 8 nt in length
» Extra SERINES at the front and at the back of the sAg insert
• Core II has less non-core derived sequence
M U I U I
sAg in Core I
sAg in Core II
M
sAg in Core I
sAg in Core II
MU I U I
Engineering inserts with spacers
Nco I Bam HI Not I Eco RI Nhe I Xho ISac I Sal I
Space(r) Shuttle
In vitro Assays
------------
ELISASPR
Future work
• Cloning remaining constructs into Tac Tandem Core vector
• Improving antigen and core independent folding– Orientation of inserts (core I or core II)– Engineering spacers for antigen sequences– Variations of insert sequences
• Development of in vitro screen– SPR– ELISA
• Yeast (?)