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Latvian Biomedical Research and Study Centre (BMC), Riga, Latvia

Latvian Biomedical Research and Study Centre (BMC), Riga, Latvia. 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.

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Latvian Biomedical Research and Study Centre (BMC), Riga, Latvia

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  1. Latvian Biomedical Research and Study Centre (BMC), Riga, Latvia

  2. 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

  3. WP1 - part I - tandem core Comparison of CoHe constructs carrying different HA stalk fragments All except No5 and No6 produces product of corresponding Mw

  4. 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

  5. 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

  6. WP1 - part I - tandem core Purification of KM71HpHe7K1,K1 (codon-optimized) from P.pastoris Immunodiffusion (+) Survives AmS precipitation Confirmed VLP formation

  7. 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

  8. WP1 - part II – single gene expression Evaluation of new M2e constructs 1 176 G4SG4G4SG4 SLLTEVETPIRNEWGCRCNDSSD TEVETPIRNEWGSRSNDSSD

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

  10. 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 M2e linker Chemical coupling as a backup strategy HA2.3needed for coupling as a single protein

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

  12. WP1 - part II – single gene expression Expression and purification of HBc176_K1VLPs 1 176 G4SG4SGGS K GGSG4SG4S tº treatment AmS precipitation S500 chromatography

  13. WP1 - part II – single gene expression Chemical coupling of HA2.3to HBc_K1VLPs + + HS- -NH2 Sulfo-KMUS S- -NH

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

  15. Our group Dr.biol. Kaspars Tars Dr.biol. Andris Kazaks Dr.biol. Tatjana Voronkova Svetlana Kotelovica InaraAkopjana Special thanks for electron microscopy pictures: Dr. VeltaOse

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