1. Protein Purification for Crystallization Dr Muhammad Imran Forman Christian College (A Chartered University) Dr Muhammad Imran Forman Christian College (A Chartered University) 1

2. Content Why we need to purify proteins? Sample requirement for crystallization. Sources of proteins for purification. Principles of different purifications strategies. Purification of un-tagged proteins (CIPP). Purification of tagged proteins. Multi-step purification. Challenges and solutions. Why we need to purify proteins? Sample requirement for crystallization. Sources of proteins for purification. Principles of different purifications strategies. Purification of un-tagged proteins (CIPP). Purification of tagged proteins. Multi-step purification. Challenges and solutions. 2

3. Why we need protein purification Total cell lysate 3 After IMAC Anion-ExchangeAfter SEC

4. Sample requirement for Crystallization Crystallization typically requires 10 mg/mL 20-50 mg/ml 10 mg/ml 2-5 mg/ml http://people.mbi.ucla.edu/sawaya/m230d/Crystallization/tipsandtricks.html BSA Quantification Gel µg/µl/lane 1 2 4 8 M 75 kDa 50 kDa 4

5. Sources of Protein 1- Wild Problems 1- Protein yield is low 2- Multiple steps for purification 3- Variation 4- Difficult to modify Advantage 1- Natural host 2- Post translational modification 3- Cloning is not required 5

6. Sources of Protein 2- Recombinant Problems 1- Post translational modification 2- Gene cloning required 3- Un-natural expression host Advantage 1- High Yielding 2- Purification tags. 3- Amenable to protein modification 6

7. Properties of proteins which we can exploit for purification Solubility…………. Salting out (Ammonium Sulphate PPT) Charge…………….. Ion Exchange Chromatography (IEC) Hydrophobicity… Hydrophobic Interaction Chromatography (HIC)HIC Affinity……………. Metal affinity, Lectin etc. Size…………………. Size Exclusion Chromatography (SEC) Tags: HIS tag……..Immobilized Metal ion Affinity Chromatography (IMAC) MBP tag…… Dextrin affinity STREP tag…. Strep-Tactin……and many other antibody affinity matrixes Solubility…………. Salting out (Ammonium Sulphate PPT) Charge…………….. Ion Exchange Chromatography (IEC) Hydrophobicity… Hydrophobic Interaction Chromatography (HIC)HIC Affinity……………. Metal affinity, Lectin etc. Size…………………. Size Exclusion Chromatography (SEC) Tags: HIS tag……..Immobilized Metal ion Affinity Chromatography (IMAC) MBP tag…… Dextrin affinity STREP tag…. Strep-Tactin……and many other antibody affinity matrixes 7

8. CIPP Purification Strategy 8

9. Purification of Tagged Proteins 9

10. 6X His Protein purification step wise Cell harvesting Cell breakage in lysis buffer Separation of debris from the cellular contents Filtration of the cell lysate Cell lysate incubation with resin 2-4 hr/over night Passing the slurry from the gravity column/HisTrap Washing the resin protein complex with wash buffer (few time) Elution using elution buffer Analysis by SDS PAGE Cell harvesting Cell breakage in lysis buffer Separation of debris from the cellular contents Filtration of the cell lysate Cell lysate incubation with resin 2-4 hr/over night Passing the slurry from the gravity column/HisTrap Washing the resin protein complex with wash buffer (few time) Elution using elution buffer Analysis by SDS PAGE 10

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12. Selection of Buffers Lysis buffer: Buffer pH, Salt concentration, stabilizer pH……Below or above Isoelectric point pI NaCl….. 100-300 mM Glycerol……5-15 %,…CPI, mild detergent, βME Washing Buffer: Lysis buffer + imidazole (1-40 mM) varies from protein to protein Elution Buffer: Lysis + 100-300 mM imidazole Lysis buffer: Buffer pH, Salt concentration, stabilizer pH……Below or above Isoelectric point pI NaCl….. 100-300 mM Glycerol……5-15 %,…CPI, mild detergent, βME Washing Buffer: Lysis buffer + imidazole (1-40 mM) varies from protein to protein Elution Buffer: Lysis + 100-300 mM imidazole 12

13. Cautions related to buffer composition HEPES interferes with the Lowry protein assay Phosphates buffers are incompatible with the use of divalent cations (e.g. Mg 2+ ions) DTT, βME, EDTA, Ionic detergents, incompatible with Ni-NTA above certain concentration. Check supplier information before preparing any buffer. HEPES interferes with the Lowry protein assay Phosphates buffers are incompatible with the use of divalent cations (e.g. Mg 2+ ions) DTT, βME, EDTA, Ionic detergents, incompatible with Ni-NTA above certain concentration. Check supplier information before preparing any buffer. 13

14. SDS-PAGE Analysis after IMAC Further Purification needed…..? 14

15. SDS-PAGE Analysis after IMAC (Another target) Do we need to further purify? 15

16. -------------------- -------------------------- -----------Cation-------------- ------------Exchanger---------- ---------Stationary Phase----- -----------Particle-------------- ---------------------------------- ---------------------------- ------------------ _ + Negatively charged analyst (Anion), adsorbed to positive surface Positively charged analyst (Cation), adsorbed to negative surface Ion Exchange Chromatography (IEC) ++++++++++++ ++++++++++++++++++ ++++++++++Anion+++++++++++ ++++++++++Exchanger++++++++++++ +++++++++Stationary phase++++++++ ++++++++++Particle+++++++++++ ++++++++++++++++++++ ++++++++++++++++ ++++++++++ 16

17. Importance of pI and salt in the sample 17

18. IEC Chemistry Several resins and beads 18

19. Steps in Ion Exchange Chromatography Equilibration of resin/column Sample application Washing Elution Regeneration Storage Equilibration of resin/column Sample application Washing Elution Regeneration Storage 19

20. A Typical IEC Chromatogram 20

21. Analysis after anion Exchange 225 150 100 75 50 35 25 15 10 Column F.T M A8 A12 B2 B4 B6 B8 B10 E2 E3 E4 E5 E6 F2 F3 WT protein sequence 21 IEC

22. Analysis after SEC: a necessary step 3 runs of WT protein sequence SEC 22

23. HIC 23

24. HIC, Role of water 24 Hydrophobic interaction depends on the behavior of the water molecules rather than on direct attraction between the hydrophobic molecules

25. HIC, Protein Structure enforced hydrophobic interaction ionic strength, the presence of organic solvents, temperature and pH (especially at the isoelectric point, pI, when there is no net surface charge) can all affect protein structure and solubility and, consequently, the interaction with other hydrophobic surfaces, such as those in HIC media. typically 1–2 M ammonium sulfate or 3 M NaCl 25

26. Challenges and how to address Challenges Expensive equipment Solution Use manual methods Use smart target Centralized facility Collaborations 26

27. Acknowledgements 27 Dr Muhammad Saleem University of Nottingham Dr Abbas Maqbool John Innes center UK

28. 28 ThanksCommentsQuestions