1. Homotandem Core-HA (and Core-GFP) Expression in Yeast Malcolm Stratford & Hazel Steels MOLOGIC

2. Contents Background Background pH/salt effects on solubility pH/salt effects on solubility Extraction methods for Core-HA Extraction methods for Core-HA Agitation effects on Core-HA Agitation effects on Core-HA Antioxidants and protease inhibitors Antioxidants and protease inhibitors Yeast Nitrogen Base media effects Yeast Nitrogen Base media effects

3. CoHo7e-GFP formation in P. pastoris Induction by methanol, optimised at 0.7% daily Induction by methanol, optimised at 0.7% daily Optimal induction in highly aerated culture Optimal induction in highly aerated culture Cultures progressively turn green, after 3 days, progressively increasing to 10 days Cultures progressively turn green, after 3 days, progressively increasing to 10 days

4. CoHo7e-GFP in P. pastoris, 5 days induction

6. Tris/pH matrix Water extraction

7. Salt/pH effect on Core-GFP solubility Starting with a large quantity of soluble Core-GFP, pH 8 in water. Added to matrix of pH 6.0-8.0, NaCl 0 – 160mM, mixed for 1.5 hours. Green fluorescence measured in total, soluble and insoluble fractions.

8. Salt/pH matrix effect on re- precipitating Core-GFP % insoluble

9. Salt/pH effect on Solubility of Core-GFP Dissolving Core-GFP required high pH and low ionic strength. This effect is fully reversible, by lowering pH and raising ionic strength It looks as if VLPs are a half-way stage between solubility and insolubility

10. Induction and extraction of Core-HA

11. Time Course E1 Core-GFP 0 1 2 3 5 7 9 11 days

12. Time Course 655 = Core-HA 0 1 2 3 5 7 9 11 days Control 654 655 strain

13. Time Course 655 = Core-HA 0 1 2 3 5 7 9 11 days x1 X5 X25 x125

14. Time Course 655 Core-HA

15. Core-HA - Extraction Methods Freeze/Thaw does this damage Core- HA? Freeze/Thaw does this damage Core- HA? Dissolving out Core-HA – does this need more work? Dissolving out Core-HA – does this need more work? Will sonication help? Will sonication help?

16. Time Course of Induction of Core-HA at different agitation rates Normally faster agitation results in faster protein production, but this will cause increased oxidative stress

17. GFP induction in P.pastoris; effect of aeration

18. Core-HA affected by Agitation RPM

19. Antioxidants BHA – Butylated Hydroxy Anisole BHA – Butylated Hydroxy Anisole Ascorbic acid Ascorbic acid EDTA EDTA No discernable effect on protein production (BHA is toxic) No discernable effect on protein production (BHA is toxic)

20. Protease Inhibitors Protease Inhibitor General (Sigma) Protease Inhibitor General (Sigma) Protease Inhibitor Fungal (Sigma) Protease Inhibitor Fungal (Sigma) Z-Leu-Leu-norvalinal Z-Leu-Leu-norvalinal Calpain Inhibitor 1 Calpain Inhibitor 1 No discernable effect on protein production (Fungal Protease is toxic) No discernable effect on protein production (Fungal Protease is toxic)

21. Yeast Nitrogen Base

22. Core-HA affected by Agitation RPM

23. Yeast Nitrogen Base, salts single excess additions, Day 7 Dilution

24. Nitrogen Sources, single excess additions, Day 7

25. Vitamins, single excess additions, Day 7

26. Mixtured nutrients excess additions, Day 7

27. Conclusions so far Nitrogen sources such as glycine seem important Nitrogen sources such as glycine seem important Vitamins seem important, but little effect when added individually – multi-vitamin effect? Vitamins seem important, but little effect when added individually – multi-vitamin effect? Yeast Extract has a positive effect – vitamins? Yeast Extract has a positive effect – vitamins? Phosphates are antagonistic Phosphates are antagonistic

28. Optimised Concentrations Glycine - 0.3g/l Glycine - 0.3g/l Vitamin mixture (x1000) – 100ul (30ul normal) Vitamin mixture (x1000) – 100ul (30ul normal) Yeast Extract – 4.5g/l Yeast Extract – 4.5g/l Yeast Nitrogen Base – 7g/l (6.7g/l normal) Yeast Nitrogen Base – 7g/l (6.7g/l normal)

29. Time Courses – improved induction media

30. Future Work Optimize Core-HA Optimize Core-HA Which vitamins? Drop-out experiments Which vitamins? Drop-out experiments Combination matrix vitamins/glycine Combination matrix vitamins/glycine Temperature effect, pH effects Temperature effect, pH effects Run Core-HA on gels Run Core-HA on gels Purify, look for VLPs Purify, look for VLPs