1. TSB Q7 Meeting 02-Jun-2009 Hepatacore iQur Leeds Progress

2. Overview Tandem core production »HA-tandem trials and tribulations Dual tandem production »eGFP-tandem »sAg,eGFP-dual tandem Space(r) Tandem core »HAVP1-tandem core »sAg-tandem core Future work

3. Bacteria expression Sizes of the tandems discussed: CoHo7e, HA1s(67kDa) CoHo7e, eGFP(64kDa) CoHo7sAg, eGFP(69kDa) CoHo7e, sAg (spacers)(~43kDa) CoHo7e, HAVP1 (spacers) (~128kDa)

4. Batch 7 HA – Tandem core purification Discontinuous sucrose gradient Load Pellet 2345678910111230 M BottomTop 1ml fractions 40%60% 30% Load Pellet 23457891011126 Biotin Mr Cores band at the 60:40% interface

5. Making HA-tandem… or not Adapted from the novel coHo7sAg purification method (see Q6 presentation), a similar high pH – low pH approach was attempted with HA-tandems Also attempted higher pH, lower buffer concentration Removal of incompatible reagents (Tween 20 precipitates with AmSO 4 ) Increasing induction temperature to decrease heavy sediments (16 raised to 27 o C)

6. An example of how NOT to do it… 23456789101131PM BottomTop 1ml fractions 40%60% 30% Eden cell pellet 16 o C induction High pH lysis (pH8.5) => Heavy sediment 234567891011PLoadM BottomMiddle 1ml fractions 40%60% iQur cell pellet 27 o C induction Normal pH lysis (pH7.5) => Lower overall yield 67kDa

7. HA-Tandem (+ fiends) shipped HA-tandem S/NP M

8. Lessons learnt for HA-tandem High pH adversely affects HA1 tandems core sedimentation Altered morphology can not be recovered by lowering pH Tween 20 is recommended at the start (post lysis) to prevent aggregates Low temperature expression levels yield better final product »(less co-purifying ~37kDa contaminant, which is probably further reduced by differential precipitation with Ammonium Sulphate)

9. Green Dual tandem - Intro Dual construct needed to show “USP” eGFP correctly folded fluorescent activity present Easy to detect Both HBsAg and eGFP inserts have previously been shown to elicit immune responses Can be used for preliminary studies in non human primates

10. Dual tandem cloning pET28b-coHe7sAg,eGFP Core II (aa1-185) pET28b-CoHe7e His eGFP Core I (aa1-149) NcoIBam HINot IEco RIXhoI Sac ISal I Flexible linker Nhe Core II (aa1-185) pET28b-CoHe7e,eGFP His Hetero tandem NheI pET28b-CoHo7sAg,e His sAg NcoIBam HINot IEco RIXhoI Sac I Sal I Flexible linker pET28b- His Digest EcoRI/NheI Homo tandem

11.  Expression not optimal at 37 ˚C MMNI M M I I I I I I 75kDa 25kDa pET coHo7e pET coHo7e, eGFP pET coHo7sAg, eGFP pET coHo7e pET coHo7e, eGFP pET coHo7sAg,eGFP 75kDa 25kDa 75kDa 25kDa Small scale expression Growth to an OD of 0.7 at 37˚C Induction with 1mM IPTG for 4h at 37˚C

12. 60 40 30 cores Analyses: Bradford SDS-PAGE Western blot ELISA TEM IPTG French Press 14000 psi Crude Dual tandem core prep 16 o C Sonication Lysis in Tris pH8, 5% glycerol, 5mM DTT, Prot. Inhib, benzonase : Clarification: 26k x g spin  Soluble  Insoluble 2 passes Quick Pellet 30% sucrose + 150mM NaCl

13. pET coHo7e,eGFP pET coHo7sAg,eGFP MNII M I M: Marker; NI: Non Induced; I: Induced Expression M T T M I S I S 250 150 100 75 50 37 25 20 10 15 pET coHo7e,eGFP pET coHo7sAg, eGFP M: Marker; T: Total Lysate; I: Insoluble Lysate; S: Soluble Lysate Solubility  Most of the protein is soluble Large scale prep (at 16 o C) 250 150 100 75 50 250 150 100 75 50

14. M : Marker; L : Load; S/N : Supernatant; P : Pellet M L L M S/N P P pETcoHo7e, eGFP pETcoHo7sAg, eGFP 250 100 75 50 150 37 25 20 10 15 L M S/N P P 250 100 75 50 150 37 25 20 10 15 M L pETcoHo7e, eGFP pETcoHo7sAg, eGFP Western blot: 10E11 dil.1/4000; Goat anti mouse dil.1/2000 M: Marker; L: Load; S/N: Supernatant; P: Pellet Particulate material inferred by sedimentation

15. Discontinuous Sucrose Gradient Bradford assay

16. M789101112LP561314 coHo7sAg, eGFP M789101112LP561314 coHo7e, eGFP Discontinuous Sucrose Gradient SDS-PAGE Coomassie stained gels

17. Broad range of sedimentation for both tandems M25791112141822263034 60%40%30% 1ml fractions BottomTopBottom coHo7sAg, eGFP (69kDa) 250 150 100 75 50 37 25 20 15 M25791113141822263430 1ml fractions 60%40% 30% TopBottom coHo7e, eGFP (64kDa) 250 150 100 75 50 37 25 20

18. Antigenicity of gradient fractions e, eGFP (1)

19. Antigenicity of gradient fractions sAg,eGFP (2)

20. Preparation of pooled fractions e, eGFPsAg, eGFP 50 37 150 250 25 75 20 100 15 10 S/NP M 50 37 150 250 25 75 20 100 15 10 S/NP M Sucrose cushion pellet represents final preparation Formulated in Tris (20mM, pH8) buffer with salt (150mM) and 40% sucrose Major co-purifying contaminant at ~37kDa

21. sAg and HAVP1 space(r) types Core I : atctggaaggatccgccggcggcggccgcgatccgg L E G S A G G G R D P A BamHI Core II: gggtcggcaataatctggaattcgccggcgctagcgac EcoRI NheI N L E F A G A S D P A S NotI Antigen insert site II Nhe IEco RI HAVP1 Afl II TYPE A Antigen insert site II Nhe IEco RI HAVP1 KpnI TYPE C Antigen insert site II Nhe IEco RI HAVP1 KpnIAfl II TYPE B Antigen insert site II Nhe IEco RI sAg Afl II Antigen insert site II Nhe IEco RI sAg KpnI Antigen insert site II Nhe IEco RI sAg KpnIAfl II

22. 16C Unind Type A 30C O/N Type A 16C Unind Type C30C Unind Type A 30C Unind Type B 30C Unind Type C 30C O/n Type C Marker16C Unind Type B16c O/N Type B 16C O/N Type C16C O/N Type A30C O/N Type B 250 150 100 75 50 37 25 20 128 kDa Blot of HAVP1 Spacers expression at 16°C and 30°C Type A gives better expression than type B Type B produces more lower order species Type C least well expressed 16 o C (weak but visible) better than 30 o C (very faint) in all cases

23. 150KDa 100KDa 16C Total Marker 16C Soluble 16C Insoluble 16C Total 16C Soluble 16C Insoluble 16C Soluble 16C Insoluble Type AType BType C *** * Sample not correctly normalised (15-fold conc) HAVP1 space(r) solubility shows little variation Solubility [more > less] follows [C > A > B] Types A and C appear slightly more soluble than type B Type A more abundant than the others Difficult to assess due to concentrated insoluble fraction and over exposure of blot

24. sAg spacer expression MU 4 hrsO/N UU 4 hrsO/N 4 hrs M 50 KDa 37 KDa Expression at 16 degree Celsius 42 KDa Type AType BType C

25. Elute immobilised fraction VLPs Immunoaffinity purification Possible generic purification approach capturing with anti-core Select for correctly folded inserts using conformation-dependent antibodies Typically low yields Very expensive Dependent on good reagents Wash off non-specific contaminants HCP Load sampleImmobilise antibody

26. Future Work Optimise purification of dual tandem cores Set up immunoaffinity column Further expression and purification of space(r) constructs In vitro assays »Polyclonal anti-HAV »Alternative anti-HBsAg Engineering 4-helix bundle (?)