1. TSB Meeting 4 Hepatacore iQur Leeds Progress

2. Overview Introduction CoHo7e,HA1s VLP purification Cloning Yeast cell lysis Future work

3. The tandem core platform Core I (aa1-149) Nco IBam HINot IEco RIXho I Sac ISal 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 protein Flexible linker

4. Target Pathogens Hepatitis B virus Enveloped virus Neutralising antigen surface antigen (HBsAg, aa124-137) Current vaccine – yeast expressed HBsAg VLPs 10 KDa insert 108 155 Core ICore II Nco I Bam HINot IEco RINhe IXho ISac ISal I HBsAg (108-155) Flexible linker Antigen insert site I Antigen insert site II

5. Target Pathogens Hepatitis A virus VP4 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 ICore II Nco I Bam HINot IEco RINhe IXho ISac ISal I HAV P1 (aa1-791) Flexible linker Antigen insert site I VP4 VP2 VP3 VP1 135 KDa

6. Influenza Haemagglutinin C2PR8HA_F2 C2PR8HA_R2 C2PR8HA_F1 C2PR8HA_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

7. 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. 182 116 82 64 49 37 26 19 15 3 6 912 15 18212427303336 M Fractions collected from the bottom of the gradient

8. 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

9. Final purification method 1.Lysis French 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 lysate Overnight incubation, protein and centrifugation at 26,000 x ‘g’ for 30 min 3.Discontinuous sucrose gradient 30%, 40% and 60% sucrose in 20mM Tris HCl pH7.0 or pH8.0, 250 mM NaCl 4.Concentration and buffer exchange Centriprep 10KDa filter

10. Analysis of CoHo7e,HA1s VLPs MI546464 MI546 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.

11. Analysis of CoHo7e,HA1s VLPs 2M1 CoHo7e,HA1s SDS PAGE analysis of CoHo7e,HA1s final sample. M. Mw markers,1. 1 μl sample, 0.5 μl sample. M11M CoHo7e,HA1s AB Western blot analysis of CoHo7e,HA1s final sample. Gels. A: Anti-HBcAg (10E11); B: Anti-His tag. M. Mw markers, 1. final sample

12. Analysis of CoHo7e,HA1s VLPs Anti-HBcAg (10E11) and anti-PR8 ELISA analysis serially diluted CoHo7e,HA1s final sample EM analysis of CoHo7e,HA1s final sample

13. Analysis of CoHo7e,HA1s VLPs Haemagglutinantion assay of CoHo7e,HA1s Samples 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

14. 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).

15. 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

16. 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!!!

17. 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.

18. 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

19. Cloning Troubleshooting 30603060 RT14 o C mins No Ligase Uncut M + Ligase

20. 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.

21. Cloning Troubleshooting Cut vector – Ligase Cut vector + Ligase Gel purifiedNot Gel purified 4 colonies 20 colonies ~180 colonies >200 colonies

22. 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.

23. Alternative yeast vector

24. Cloning pPIC3.5K-CoHo7e Clone into pPIC3.5KCoHo-R1_3’ (XcmI/EcoRI digested)

25. Cloning pPIC3.5K-CoHo7e ~450bp ~250bp coHo7e, e coHo7e, sAg

26. 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

27. 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

28. Yeast cell lysis SDS PAGE and western blot (anti-core) analysis of yeast cell lysis (CoHBc149 and CoHo7e,eGFP expression) M TS I CoHo7e eGFP TS I CoHBc149 MTS I CoHo7e eGFP TS I CoHBc149

29. Yeast cell lysis Western blot (anti-core) analysis of yeast cell lysates - bead beater lysis CoHo7e,eGFP E1 CoHo7e,HA1s F1 HBc149 B1 HBc149 B2 CoHo7e,HA1s F2 CoHo7e A1 CoHo7e C1 CoHo7e D1

30. 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

31. 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 !

32. 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 TS I + Tween 20 TS I -Tween 20 M TS I + Tween 20 TS I -Tween 20 sAg in Core II

33. 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