Report summary
Testing of HA stalk constructs
Testing of M2e constructs
Testing of K1K1 construct
Evaluation of new M2e constructs
Evaluation of new HA stalk constructs
Evaluation of long homodimer constructs

WP1 - part I - tandem core
Comparison of CoHe constructs carrying different HA stalk fragments
No Construct Mw, kDa
1 pET coHe7HA1(306-351), empty
2 pET coHe7HA2(306-402), empty
3 pET coHe7HA3(403-474), empty
4 pET coHe7LAH(420-474), empty
5 pET coHe7∆HAStalk,empty
6 pET coHe7empty, Headless HA
7 pET coHe7empty, HA2(306-402)
8 pET coHe7empty, HA4(296-402)
9 pET coHe7empty, LAH(420-474)
46.8
52.3
50.2
49.4
77.9
78.0
52.4
~55.0
49.5
All except No5 and No6 produces product of corresponding Mw

WP1 - part I - tandem core
Solubility of recombinant HBc-HA proteins
At least part of protein is in soluble fraction
No5 is insoluble. No6 is degraded
No detectable immunodiffusion bands, except No6

WP1 - part I - tandem core
Synthesis and solubility of recombinant HBc-M2e proteins
No Construct
1 pETcoHe7e,M2eSP
2 pETcoHe7e,M2e
C19S
3 pETcoHe7e,M2e
C17,19S
4 pETcoHe7e,M2e
C17,19S
SP
5 pETcoHe7(M2e
C17,19S
)
3
SP
Minor part of protein is in soluble fraction
No detectable immunodiffusion bands, except No5; confirmed VLP formation

WP1 - part I - tandem core
Purification of KM71H pHe7 K1,K1 (codon-optimized) from P.pastoris
Immunodiffusion (+)
Survives AmS precipitation
Confirmed VLP formation

WP1 - part II – single gene expression
Testing of HA stalk constructs
Testing of M2e constructs
Testing of K1K1 construct
Evaluation of new M2e constructs
Evaluation of new HA stalk constructs
Evaluation of long homodimer constructs

WP1 - part II – single gene expression
Evaluation of new M2e constructs
1
G
4
SG
4
G
4
SG
4
S
S
176

WP1 - part II – single gene expression
Evaluation of new M2e constructs
S
S
S
S
tº treatment AmS precipitation S500 chromatography
VLPs for immunization?

WP1 - part II – single gene expression
Evaluation of new HA stalk constructs
HA2.3 (403-474) as the most prospective immunologically
No VLPs detected by expression in tandem core
HA3
K linker
M2e
Chemical coupling as a backup strategy
HA2.3 needed for coupling as a single protein

WP1 - part II – single gene expression
Expression and purification of HA2.3 as a single protein
6His
MBP
TEV
HA3 -
Cys
HA2.3 trimer?
SEC profile
1. Expression 3-4. TEV cleavage
2. Ni-affinity 5.-7. AEC aggregates and VLPs
HA3 monomer

WP1 - part II – single gene expression
Expression and purification of HBc176_K1 VLPs
1
G
4
SG
4
SGGS K GGSG
4
SG
4
S tº treatment AmS precipitation S500 chromatography
176

WP1 - part II – single gene expression
Chemical coupling of HA2.3 to HBc_K1 VLPs
-NH
2
+
Sulfo-KMUS
+
HS-
S-
-NH

WP1 - part II
Design of long homodimer construct
HA2.3
K linker
M2e
1 176 1 176
GGSx1→GGSx3→GGSx5
No detectable expression in E. coli
GGSx6→GGSx9
Constructs in progress

Dr.biol. Tatjana Voronkova
Our group
Dr.biol. Kaspars Tars
Dr.biol. Andris Kazaks
Svetlana Kotelovica Inara Akopjana
Special thanks for electron microscopy pictures: Dr. Velta Ose