1. coHo7 Core-sAg,e, Core-sAg,HAVP (and Core-HA, Core-GFP) Expression in Yeast Malcolm Stratford & Hazel Steels MOLOGIC

2. Contents Background Background Optimization of Core-HA Optimization of Core-HA Yeast strain effects Yeast strain effects Transformation sAg,e and sAg,HAVP Transformation sAg,e and sAg,HAVP Optimizing sAg,e and sAg,HAVP Optimizing sAg,e and sAg,HAVP Western blot effects Western blot effects

3. Green – 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. GFP induction in P.pastoris; effect of aeration

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

8. Core-HA

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

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

11. Time Course 655 Core-HA

12. 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)

13. Time Courses – improved induction media

14. Combinations of Vitamins and Glycine – Core-HA 7 day induction Optimum 1g/l glycine + x10 vitamins

15. Induction Temperature Core-HA Induction + x10 vitamins

16. Induction pH on Core-HA Relatively small effect over the range pH 3.5 – pH 7.0 Relatively small effect over the range pH 3.5 – pH 7.0 pH optimum 4-5 pH optimum 4-5 Water wash pre-induction reduced extraction, removing vitamins form yeast extract Water wash pre-induction reduced extraction, removing vitamins form yeast extract 3 days induction, media standard + x10 vitamins

17. P. pastoris strains X33 – used for Core-GFP - 2 days for growth SHD 1168 – used for Core-HA - protease deleted - 3 days for growth

18. Transformation sAg,e and sAg,HAVP Supplied on pPIC 3.5 vector Supplied on pPIC 3.5 vector G418/kanamycin selection in yeast G418/kanamycin selection in yeast Vector linearized using Pma1 Vector linearized using Pma1 First attempt – transformation and direct selection on G418 First attempt – transformation and direct selection on G418 Zero transformants selected Zero transformants selected

19. 2 nd attempt - Transformation sAg,e and sAg,HAVP pPIC 3.5 vector, primary selection using His auxotrophy, secondary selection using G418 pPIC 3.5 vector, primary selection using His auxotrophy, secondary selection using G418 His auxotophy gives many false positives His auxotophy gives many false positives 1000’s of transformants pooled and then plated out on increasing G418 1000’s of transformants pooled and then plated out on increasing G418

20. 2 nd attempt - Transformation sAg,e and sAg,HAVP 20 transformants of each selected

21. Selecting the best transformants 2 days induction, Standard induction + x10 vitamins Select sAg,e 10, 20 HAVP 30, 38

22. Time Courses sAg,e and,HAVP Standard induction + x10 vitamins

23. Varying Methanol Methanol added daily as AOX inducer Methanol added daily as AOX inducer Used up by growing yeast Used up by growing yeast Toxic in high levels Toxic in high levels 0.7% optimized level for Core-GFP in strain X33 0.7% optimized level for Core-GFP in strain X33 Strain SHD 1168 grows slower does it use methanol slower? Strain SHD 1168 grows slower does it use methanol slower?

24. Time Course 655 Core-HA 0.7% methanol daily

25. Varying Methanol Strain sAg,e 20

26. Bulk production 800ml cultures sAg,e 20 and,HAVP 30 800ml cultures sAg,e 20 and,HAVP 30 3 day induction 3 day induction Methanol 0.5% Methanol 0.5% Induction media + x10 vitamins Induction media + x10 vitamins Yielded ~ 50mls extract, glass beads and cell debris removed Yielded ~ 50mls extract, glass beads and cell debris removed Sigma Protease Inhibitor Cocktail P8215 (fungal)1ml to 100mls Sigma Protease Inhibitor Cocktail P8215 (fungal)1ml to 100mls Frozen and sent to Aadil Frozen and sent to Aadil

27. Western blot sAg,e and sAg,HAVP 3 days induction, Standard induction media + x10 vitamins 188 98 64 49 38 28 17,e,e/10,H,H/10 sAg,e 42kDa HAVP 130kDa

28. Odd Bands in Western Blot Only in neat samples, not in X10 water dilution Only in neat samples, not in X10 water dilution Both from same frozen samples Both from same frozen samples Both centrifuged and heated with SDS for gel Both centrifuged and heated with SDS for gel Diluted ones contained less Core, cell extract, buffer (succinate) and Protease inhibitor cocktail. Diluted ones contained less Core, cell extract, buffer (succinate) and Protease inhibitor cocktail. Suggest one of these is causing Core break-up in SDS heat stage Suggest one of these is causing Core break-up in SDS heat stage