1. Heterologous Protein Expression in Yeast CoHo7e - Green, Core and HA Malcolm Stratford & Hazel Steels MOLOGIC

2. Contents Introduction Introduction Core+GFP expression in Pichia pastoris Core+GFP expression in Pichia pastoris Sonication Sonication CTAB CTAB Protein gels Protein gels Protein measurement Protein measurement Antibody detection Antibody detection

3. Yeast expression system Pichia pastoris, integrating vector, zeocin selection, AOX promoter induced by methanol Pichia pastoris, integrating vector, zeocin selection, AOX promoter induced by methanol

4. 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

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

6. Green – Physical Extraction Grinding with glass beads yields a green slurry Grinding with glass beads yields a green slurry Hard centrifugation showed almost all green fluorescence in the pelleted fraction Hard centrifugation showed almost all green fluorescence in the pelleted fraction However, there was an intense green fluorescent layer at the top of the pellet However, there was an intense green fluorescent layer at the top of the pellet

9. Green Slurry Contains yeast cell debris and ~ 10% GFP in solid particles Contains yeast cell debris and ~ 10% GFP in solid particles Green particles appear damaged by grinding but remain substantially intact Green particles appear damaged by grinding but remain substantially intact No evidence of membranes around particles – look like solid protein inclusion bodies. No evidence of membranes around particles – look like solid protein inclusion bodies. Without fluorescence, green particles indistinguishable from other debris Without fluorescence, green particles indistinguishable from other debris

10. Tris/pH matrix Water extraction

11. pH adjustment without buffers Water extraction

12. Extracting CoHo7e-Green Most ~ 60% - 80% Green is made soluble by raising the pH to 8.0 in low ionic strength. Most ~ 60% - 80% Green is made soluble by raising the pH to 8.0 in low ionic strength. Can we use sonication or CTAB to extract the remainder? Can we use sonication or CTAB to extract the remainder? Can we get a reliable band on a protein gel? Can we get a reliable band on a protein gel?

13. Sonication Sonicating water bath – no effect Sonicating water bath – no effect Probe sonication (MSE –Soniprep) Strong effect but also caused strong local heating effects Probe sonication (MSE –Soniprep) Strong effect but also caused strong local heating effects How much heat will CoHo7e-GFP stand? How much heat will CoHo7e-GFP stand? Is the effect of sonication via heat or ultrasonics? Is the effect of sonication via heat or ultrasonics?

14. Heat Resistance of Core-Green Green supernatants, pH 8.1 in water, 10 mins at each temperature

15. Sonication - heat 30ml volume, 10mm probe, start = 16C

16. Sonication – extraction of pellets 30ml volume, 10mm probe, start = 16C

17. Sonication CoHo7e-GFP is heat stable, 10 mins at 60C CoHo7e-GFP is heat stable, 10 mins at 60C Probe sonication caused strong local heating effects, 50C over 1 minute Probe sonication caused strong local heating effects, 50C over 1 minute Sonication caused extraction of green from pellets in < 30 secs (not a heating effect) Sonication caused extraction of green from pellets in < 30 secs (not a heating effect) Sonication had no effect on green in total preps, decreased green in pellets and increased green in soluble fractions Sonication had no effect on green in total preps, decreased green in pellets and increased green in soluble fractions

18. CTAB Previous work had showed CTAB to help solublize green at high and low concentration but not at 1mM Previous work had showed CTAB to help solublize green at high and low concentration but not at 1mM

19. CTAB – extraction of Green Water extraction, in 25mM HEPES pH 8

20. CTAB effect on Protein Gels CTAB appeared to destroy larger protein bands CTAB 0.4 4mM pH 6 pH 8 Sonic

21. Protein Gels Can we detect Core-GFP on a protein gel? Can we detect Core-GFP on a protein gel? Tested effect of protein concentration (serial dilution) Tested effect of protein concentration (serial dilution) Tested effect of prep temperature Tested effect of prep temperature Tested effect of non-reducing/ reducing Tested effect of non-reducing/ reducing Best results – reducing gels 95C for 10 mins Best results – reducing gels 95C for 10 mins

22. X33 and E1 soluble fractions X33 Control Core-GFP E1 66kDa

23. Core and Core-HA Core = 37kDa, Core-HA = 67kDa Core = 37kDa, Core-HA = 67kDa No comparable bands seen on gels No comparable bands seen on gels Tried 100C SDS treatment in reducing gels Tried 100C SDS treatment in reducing gels Tried high pH, low ionic strength Tried high pH, low ionic strength Tried CTAB Tried CTAB Tried sonication Tried sonication Are these proteins not formed, or not soluble? Need an antibody for detection.

24. Protein detection - Bradford Core – GFP is insoluble at pH 6 but soluble at pH 8. Core – GFP is insoluble at pH 6 but soluble at pH 8. It appears to be in high concentration It appears to be in high concentration Can we detect it using Bradford reagent? Can we detect it using Bradford reagent? Is there more than in the control? Is there more than in the control? Can we use this to detect Core and Core+HA ? Can we use this to detect Core and Core+HA ?

25. Detection of Core-GFP using Bradford Water extraction pH 8.0

26. Antibody Method - Detection of Core Protein Antibody to Core protein from AbCam Antibody to Core protein from AbCam Method from Aadil Method from Aadil Caution as to non-specific binding of yeast proteins Caution as to non-specific binding of yeast proteins Dot blot method established, based on Aadil’s method, calibrated used Core- HA protein from Barry Dot blot method established, based on Aadil’s method, calibrated used Core- HA protein from Barry

27. Antibody detection of core protein ControlControl Eden – HA E1 - GFP Total SolubleTot Sol Tot Sol Tot Sol X1 X5 X25 X125 x625

28. Conclusion We can detect Core protein from E1, CoHo7e-GFP, and from Eden - CoHo7e- HA We can detect Core protein from E1, CoHo7e-GFP, and from Eden - CoHo7e- HA Amounts detected ~ 1200mg/l E1, 200mg/l Eden – HA Amounts detected ~ 1200mg/l E1, 200mg/l Eden – HA Large proportion of both in the soluble fraction at pH 8.0 Large proportion of both in the soluble fraction at pH 8.0

29. Future Work Use dot-blot method to screen all transformants for production of core (i.e. tandem Core, Core, Core + GFP, Core + HA. Use dot-blot method to screen all transformants for production of core (i.e. tandem Core, Core, Core + GFP, Core + HA. Where no protein found in transformants, check vector construct and yeast insert by DNA sequencing Where no protein found in transformants, check vector construct and yeast insert by DNA sequencing Optimise production of Core-HA, as detected by dot-blot Optimise production of Core-HA, as detected by dot-blot