tsb meeting 4 hepatacore iqur leeds progress. overview introduction coho7e,ha1s vlp purification...
TRANSCRIPT
TSB Meeting 4
Hepatacore
iQur Leeds Progress
Overview
• Introduction
• CoHo7e,HA1s VLP purification
• Cloning
• Yeast cell lysis
• 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
Target Pathogens
Hepatitis B virus
• Enveloped virus
• Neutralising antigen surface antigen (HBsAg, aa124-137)
• Current vaccine – yeast expressed HBsAg VLPs
• 10 KDa insert
108155
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 II
Target PathogensHepatitis A virus
VP
4 VP2 VP3 VP1
HAV P1
• Non-enveloped virus
• Neutralising antigen – cluster of epitopes in VP1 and VP3
• Current vaccines – live attenuated or inactivated whole virus
• 100 KDa insert
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
135 KDa
Influenza Haemagglutinin
C2PR8HA_F2C2PR8HA_R2
C2PR8HA_F1C2PR8HA_R1
• 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 and biophysical (EM, CD) assays
E.Coli expression and purification of CoHo7e,HA1s
• Resuspended sucrose cushion pellet is separated on a discontinuous sucrose density gradient (20-60%).
• A contaminating E.coli band (~ 37KDa) sediments with the CoHo7e,HA1s protein.
18211682
6449
37
26
1915
3 6 9 12 15 18 21 24 27 30 33 36M
Fractions collected from the bottom of the gradient
Removing the contaminant
Modification of sucrose density gradient purification• Heat treatment of lysate• Urea treatment of lysate
Affinity purification• His-tag nickel affinity column• Fetuin pull-down
Anion exchange chromatography• +/- 2M Urea• +/- Dialysis pretreatment
Ammonium sulphate precipitation
Final purification method
1. LysisFrench press: 14,000 psi, 2 passes.Sonication: 6 cycles 10s on, 20s off, 10 micron amplitude (on ice).Tween-20 treatment: 0.05% Tween-20 incubated at room temperature on rotating mixer for 1 hr.Clarification spin: Centrifugation of lysate at 50,000 x ‘g’ for 1 hr at 8 °C Dialysis: dialysed against 50 mM Tris HCl pH8.0, 2 M urea for 24hr at
4 °C using a 10 kDa MWCO Slide-a-Lyzer (Pierce) 2. Ammonium sulphate precipitation
50% ammonium sulphate added to the dialysed cell lysateOvernight incubation, protein and centrifugation at 26,000 x ‘g’ for 30 min
3. Discontinuous sucrose gradient30%, 40% and 60% sucrose in 20mM Tris HCl pH7.0 or pH8.0, 250 mM NaCl
4. Concentration and buffer exchangeCentriprep 10KDa filter
Analysis of CoHo7e,HA1s VLPs
M I 54 64
M I 54 6
pH 7.0 pH 8.0
CoHo7e,HA1s
SDS PAGE analysis of discontinuous sucrose density gradient purification of prep VIII CoHo7e,HA1s.
M. Mw markers, I. Input (resuspended ammonium sulphate precipitated protein)4-6. Sucrose gradient fractions 4-6 from the bottom of the gradient.
Analysis of CoHo7e,HA1s VLPs
2 M1
CoHo7e,HA1s
SDS PAGE analysis of CoHo7e,HA1s final sample.M. Mw markers,1. 1 μl sample, 0.5 μl
sample.
M 1 1M
CoHo7e,HA1s
A B
Western blot analysis of CoHo7e,HA1s final sample.
Gels. A: Anti-HBcAg (10E11); B: Anti-His tag. M. Mw markers, 1. final sample
Analysis of CoHo7e,HA1s VLPs
Anti PR8 and anti HBcAg ELISA
0
0.5
1
1.5
2
2.5
1 in 5
1 in 10
1 in 20
1 in 40
1 in 80
1 in 160
1 in 320
1 in 640
1 in 1280
1 in 2560
well
Ab
so
rba
nc
e
anti-PR8
anti-HBcAg
Anti-HBcAg (10E11) and anti-PR8 ELISA analysis serially diluted
CoHo7e,HA1s final sample
EM analysis of CoHo7e,HA1s final sample
Analysis of CoHo7e,HA1s VLPs
Haemagglutinantion assay of CoHo7e,HA1sSamples incubated with chick erythrocytes for 72hr at room temperature
PBS only
coHo7e,HA1s Prep X + Tween 20
coHo7e,HA1s Prep X - Tween 20
coHo7e,HA1s Prep XI – Tween 20 coHo7e Prep I – Tween 20
Influenza PR8
Transfer of constructs to yeast expression vector
• All constructs to be used in this study must be transferred to a Pichia pastoris expression vector
• pPICZ-C was the vector selected.
• For initial studies of tandem core expression in yeast, the following vectors were prepared
1. Wt HBc149 (to compare wt sequence with E.coli optimised sequences).
2. CoHo7e (empty E.coli optimised tandem core).
3. CoHo7e,eGFP (eGFP provides a simple assay for expression levels during optimisations).
4. CoHo7e,HA1s (Haemagglutinin construct).
Transfer of constructs to yeast expression vector
Following successful cloning of these constructs, SOPs were written
and carefully followed to avoid problems in future cloning steps
Transfer of constructs to yeast expression vector
The following constructs are now being prepared:
1. CoHo7e,HAVP1 (HAVP1 sequence in core II)
2. CoHo7,sAg (HBV surface antigen in core I)
3. CoHo7sAg,HAVP1 (dual inserts- sAg in core I and HAVP1 in core II)
More problems with cloning have delayed the production of these constructs!!!
Cloning Troubleshooting
A list of steps involved in the cloning process was drawn up.
1. Plasmid purification / PCR amplification of inserts
2. Restriction digestion
3. Gel purification of insert and vector
4. Ligation
The only change from previously optimised cloning experiments was found to be the use of a new UV light box!
Could the UV light now be damaging insert and vector?
A safe blue filter was used on the UV light box to limit damage of the DNA.
Cloning Troubleshooting
The following experiment was set up to determine the efficiency of plasmid digestion and re-ligation / transformation.
1. Digest plasmid with a pair of enzymes to release an insert
2. Heat inactivate enzyme
3. Ligate for 30 min / 1 hr at RT or 14°C (+/- ligase)
4. Heat inactivate ligase at end of incubation
5. Run ligation mixes on an agarose gel to check for re-ligation
6. Transform E.coli DH5α with ligated plasmid
Result:
Ligation and transformation were successful in the presence of ligase
Cloning Troubleshooting
30 60 30 60
RT 14oC
minsNo
Liga
se
Unc
ut
M
+ Ligase
Cloning Troubleshooting
A second experiment was set up to determine the effect of gel purification on ligation / transformation.
1. Digest plasmid with a single enzyme to linearise
2. Heat inactivate enzyme and gel purify half of the reaction
3. Ligate both gel purified and non purified linearised vector (+/- ligase)
4. Transform E.coli DH5α with ligated plasmid
Result:
Ligation and transformation were successful in the presence of ligase for both gel purified and non-purified linearised vector.
Cloning Troubleshooting
Cut vector – Ligase
Cut vector + Ligase
Gel purified Not Gel purified
4colonies
20colonies
~180colonies
>200colonies
Alternative yeast vector
One factor in the cloning difficulties has been the use of the pPICZ-C vector.
Many of the useful restriction sites are found within the vector making cloning strategies complicated
Zeocin selection (bacteriostatic) has led to screening of negative clones.
Expression of the constructs has not given high yield of protein
An alternative vector pPIC 3.5K has both ampicillin and kanamycin resistance markers.
Alternative yeast vector
Cloning pPIC3.5K-CoHo7e
Core I (aa1-149)
Nco I Bam HI Not I Eco RI Kas ISac I Sal I
Flexible linker
Antigen insert site I
Antigen insert site II
Nhe I
Core II (aa1-149)
pPICZC CoHo7e
Xcm I
Kpn I
Xho I
PCR Amplify
Digest pPIC3.5K with EcoRI and Not I
Digest insert with EcoRI and Psp0MI
Eco RI Mlu IAfl IISpi I Psp0M I
Xcm I
pPIC3.5K CoHo-R1_3’
Not IEco RI Mlu IAfl II
Spi I
Psp0M I
Bam HI Not I Eco RISac I Sal IXcm I
Digest pPICZC with XcmI and EcoRI
Bam HI Not I Eco RISac I Sal I
pPIC3.5K CoHo7e
Mlu IAfl II Spi IXcm I
Clone into pPIC3.5KCoHo-R1_3’ (XcmI/EcoRI digested)
Cloning pPIC3.5K-CoHo7e
~450bp~250bp
coHo7e, e coHo7e, sAg
Yeast cell lysis
The following yeast cell pellets were obtained from Mologic
• Wt HBc149 (to compare wt sequence with E.coli optimised sequences) 18kDa.
• CoHo7e (empty E.coli optimised tandem core) 37kDa• CoHo7e,eGFP (65kDa)• CoHo7e,HA1s (67kDa)
Lysis method French press: 14,000 psi, 4 passes.Sonication: 6 cycles 10s on, 20s off, 10 micron amplitude (on ice).Tween-20 treatment: 0.05% Tween-20 incubated at room temperature on rotating mixer for 1 hr.Clarification spin: Centrifugation of lysate at 50,000 x ‘g’ for 1 hr at 8 °C
Yeast cell lysis
Western blot detection was necessary to detect any expressed protein
Only CoHo7e,eGFPs expression lysates gave positive results
It appeared that the core protein was soluble but insoluble material may not enter the gel
Glass bead vortexing did not improve lysis
PCR analysis of genomic DNA showed that each of the cell expression pellets were from transfected yeast cells
Use of a bead beater (3 min, 0.5mm beads) allowed detection of several constructs by western blot
Yeast cell lysis
SDS PAGE and western blot (anti-core) analysis of yeast cell lysis (CoHBc149 and CoHo7e,eGFP expression)
M T SI
CoHo7e eGFP
T SI
CoHBc149
MT SI
CoHo7e eGFP
T SI
CoHBc149
Yeast cell lysis
Western blot (anti-core) analysis of yeast cell lysates - bead beater lysis
Mco
Ho7
eeG
FP
HB
c14
9(B
1)
HB
c14
9(B
2)
coH
o7e,
HA
1s(F
1)
Tot Sol Tot Sol Tot Sol Tot Sol
coH
o7e,
HA
1s(F
2)
coH
o7e,
eLe
eds
(A1)
coH
o7e,
eLe
eds
(C1)
coH
o7e,
eM
olo
gic
(D1)
MTot Sol Tot Sol Tot Sol Tot SolMco
Ho7
eeG
FP
HB
c14
9(B
1)
HB
c14
9(B
2)
coH
o7e,
HA
1s(F
1)
Tot Sol Tot Sol Tot Sol Tot SolMco
Ho7
eeG
FP
HB
c14
9(B
1)
HB
c14
9(B
2)
coH
o7e,
HA
1s(F
1)
Tot Sol Tot Sol Tot Sol Tot SolMco
Ho7
eeG
FP
HB
c14
9(B
1)
HB
c14
9(B
2)
coH
o7e,
HA
1s(F
1)
Tot Sol Tot Sol Tot Sol Tot SolTot Sol Tot Sol Tot Sol Tot Sol
coH
o7e,
HA
1s(F
2)
coH
o7e,
eLe
eds
(A1)
coH
o7e,
eLe
eds
(C1)
coH
o7e,
eM
olo
gic
(D1)
MTot Sol Tot Sol Tot Sol Tot Sol
coH
o7e,
HA
1s(F
2)
coH
o7e,
eLe
eds
(A1)
coH
o7e,
eLe
eds
(C1)
coH
o7e,
eM
olo
gic
(D1)
MTot Sol Tot Sol Tot Sol Tot Sol
coH
o7e,
HA
1s(F
2)
coH
o7e,
eLe
eds
(A1)
coH
o7e,
eLe
eds
(C1)
coH
o7e,
eM
olo
gic
(D1)
MTot Sol Tot Sol Tot Sol Tot SolMTot Sol Tot Sol Tot Sol Tot Sol
CoHo7e,eGFPE1
CoHo7e,HA1sF1
HBc149B1
HBc149B2
CoHo7e,HA1sF2
CoHo7eA1
CoHo7eC1
CoHo7eD1
Expression of sAg in core I or core II (E.coli)
All current tandem core clones have foreign sequence inserted in core II
For production of a tandem core construct with two inserts, HBV surface antigen (sAg) was transferred to core I
pET28b CoHo7e was used as the parental vector
Expression of CoHo7sAg,e and CoHo7e,sAg was compared in E.coli BL21/DE3 cells
Expression of sAg in core I or core II (E.coli)
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
SDS PAGE and western blot (anti-core) analysis of CoHo7,sAg,e and CoHo7e,sAg expression in E.coli
Improved expression of sAg in core I !
Solubility of sAg in core I or core II (E.coli)
sAg in Core I
SDS PAGE analysis of CoHo7,sAg,e and CoHo7e,sAg solubility
Both proteins show good solubility
M T SI
+ Tween 20
T SI
-Tween 20
M T SI
+ Tween 20
T SI
-Tween 20
sAg in Core II
Future work
Cloning
1. Complete pPIC3.5K,CoHo7e
2. CoHo7e,sAg and CoHo7e,HAVP1 to pPIC3.5K vector (iQur/UoL)
3. Prepare dual insert construct (sAg ad HAVP1)
4. Optimisation of construct sequences for folding and solubility
Expression & purification of VLPs
1. Send above constructs pPIC3.5K constructs to Mologic for expression
2. Optimise bead mill lysis of yeast cell pellets
3. Optimise scalable purification processes