1. Protein Purification Strategies Course: Methods in protein chemistry Rahman M. Mahfuzur 2012/01/11 SLU

2. To seperate a particular protin from all other proteins and cell components There are many types of proteins within an ogranism Other components: nuclic acids, charbohydrates, lopids, small molecules A gieven protein could be 0.001-20% of total protein. Objectives

3. Proteins purification varies from one purification step to multi- step of purifixcations Often more than one purification step is necessary to reach the desired purity, or step can be repetead if sample is available. Successful and efficient protein purification depends on appropriate methods selection. Methods should be sequence in a logical manner, what kinds of materials are available/handle? what has to be removed/ completely? what will be the use of final products? what are economical constraints? Protein purification

4. Three phase purification strategies(CIPP)

5. The four parameters Every technique offers a balance between resolution, capacity, speed and recovery

6. Capture Initial purification of target Rapid isolation, stabilization, concentration

7. Intermediate purification Further removal of bulk contaminants: other proteins, nucleic acids, endotoxins and viruses. Purification and concentration.

8. Polishing Final removal of remaining trace impurities or closely related substances to achieve high purity

9. Protein propertyTechnique ChargeIon exchange (IEX) Specific ligand recognition (biospecifc or nonbiospecifc) Affinity chromatography (AC) SizeGel filtration (GF) HydrophobicityHydrophobic interaction (HIC), Reversed phase (RPC) Isoelectric pointChromatofocusing Protein properties Vs Technique

10. Ion exchange chromatography (IEX) separates proteins with differences in surface charge. based on the reversible interaction, charged protein Vs oppositely charged column If, pH b >PI P  Neg. bind  positively charged anion exchanger ; ex- MonoQ column When, pH b <PI P  Pos. bind  negatively charged cation exchanger ; ex- MonoS column

11. IEX chromatogram

12. Affinity chromatography (AC) On the basis of a reversible or specific interaction between target and a specifc ligand Biospecific: antibodies binding proteins, Non-biospecific: histidine binding protein bind to metal Ion; IMAC

13. AC chromatogram

14. Gel filtration chromatography (GF) allow separation of proteins with differences in molecular size GF is a non-binding method 0.5% to 2% of total column volume.

15. FG chromatogram

16. Hydrophobic interaction chromatography (HIC) separates proteins with differences in hydrophobicity based on the reversible interaction between a protein and the hydrophobic surface of a chromatography medium

17. Technique Vs three phase

18. combinations of chromatographic steps

19. IEX-HIC-GF Considered as a standard protocol If nothing is known about the target protein use IEX- HIC-GF. both anion and cation exchange could be used to get different selectivities within the strategy.

20. Thanks for listening me!