latvian biomedical research and study centre (bmc), riga, latvia
TRANSCRIPT
Latvian Biomedical Research and Study Centre (BMC), Riga, Latvia
Testing of HA stalk constructs
Report summary
Testing of M2e constructs
Evaluation of long homodimer constructs
Evaluation of new M2e constructs
Testing of K1K1 construct
Evaluation of new HA stalk constructs
Comparison of CoHe constructs carrying different HA stalk fragments
WP1 - part I - tandem core
No Construct Mw,
kDa
1 pET coHe7HA1(306-351), empty 46.8
2 pET coHe7HA2(306-402), empty 52.3
3 pET coHe7HA3(403-474), empty 50.2
4 pET coHe7LAH(420-474), empty 49.4
5 pET coHe7∆HAStalk,empty 77.9
6 pET coHe7empty, Headless HA 78.0
7 pET coHe7empty, HA2(306-402) 52.4
8 pET coHe7empty, HA4(296-402) ~55.0
9 pET coHe7empty, LAH(420-474) 49.5
All except No5 and No6 produces product of corresponding Mw
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
Minor part of protein is in soluble fraction
No detectable immunodiffusion bands, except No5; confirmed VLP formation
No Construct
1 pETcoHe7e,M2eSP
2 pETcoHe7e,M2eC19S
3 pETcoHe7e,M2eC17,19S
4 pETcoHe7e,M2eC17,19S SP
5 pETcoHe7(M2eC17,19S )3SP
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 I - tandem core
Testing of HA stalk constructs
Testing of M2e constructs
Evaluation of long homodimer constructs
Evaluation of new M2e constructs
Testing of K1K1 construct
Evaluation of new HA stalk constructs
WP1 - part II – single gene expression
Evaluation of new M2e constructs
WP1 - part II – single gene expression
1 176
G4SG4 G4SG4
SLLTEVETPIRNEWGCRCNDSSD TEVETPIRNEWGSRSNDSSD
Evaluation of new M2e constructs
WP1 - part II – single gene expression
TEVETPIRNEWGSRSNDSSDSLLTEVETPIRNEWGSRSNDSSD
tº treatment AmS precipitation S500 chromatography
VLPs for immunization?
Evaluation of new HA stalk constructs
WP1 - part II – single gene expression
HA2.3 (403-474) as the most prospective immunologically
No VLPs detected by expression in tandem core
Chemical coupling as a backup strategy
linkerM2e
HA3
HA2.3 needed for coupling as a single protein
K
Expression and purification of HA2.3 as a single protein
WP1 - part II – single gene expression
HA3 - CysMBP
TEV6His
1. Expression 3-4. TEV cleavage
2. Ni-affinity 5.-7. AEC
HA2.3 trimer?
SEC profile
aggregatesand VLPs
HA3 monomer
Expression and purification of HBc176_K1 VLPs
WP1 - part II – single gene expression
1 176
G4SG4SGGS K GGSG4SG4S
tº treatment AmS precipitation S500 chromatography
Chemical coupling of HA2.3 to HBc_K1 VLPs
WP1 - part II – single gene expression
-NH2
+ HS-
-NH
S-
Sulfo-KMUS
+
Design of long homodimer construct
WP1 - part II
1 176 1 176
GGSx1→GGSx3→GGSx5
No detectable expression in E. coli
GGSx6→GGSx9
Constructs in progress
linkerM2e
HA2.3
K
Our group
Dr.biol. Tatjana Voronkova
Inara Akopjana
Dr.biol. Kaspars Tars
Dr.biol. Andris Kazaks
Svetlana Kotelovica
Special thanks for electron microscopy pictures: Dr. Velta Ose