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Protein Purification Strategies

Protein Purification Strategies. Course: Methods in protein chemistry Rahman M. Mahfuz ur 2012/01/11 SLU. Objectives. To seperate a particular protin from all other proteins and cell components There are many types of proteins within an ogranism

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Protein Purification Strategies

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

  2. Objectives 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.

  3. Protein purification 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?

  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 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, pHb>PIP Neg. bind  positively charged anion exchanger ; ex- MonoQ column • When, pHb<PIP Pos. bind  negatively charged cation exchanger ; ex- MonoS column

  11. IEX chromatogram

  12. Affinity chromatography (AC) • On the basis of a reversible or specificinteraction between target and a specifcligand • 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. 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. IEX-HIC-GF

  20. Thanks for listening me!

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