Protein Purification Strategies

Course: Methods in protein chemistry Rahman M. Mahfuzur 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 Other components: nuclic acids, charbohydrates, lopids, small molecules A gieven protein could be 0.001-20% of total protein.

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?

Three phase purification strategies(CIPP)

The four parameters

Every technique offers a balance between resolution, capacity, speed and recovery

Capture

Initial purification of target Rapid isolation, stabilization, concentration

Intermediate purification

Further removal of bulk contaminants: other proteins, nucleic acids, endotoxins and viruses.

Purification and concentration.

Polishing

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

Protein properties Vs Technique

Protein property

Charge Specific ligand recognition (biospecifc or nonbiospecifc) Size Hydrophobicity Isoelectric point

Technique

Ion exchange (IEX) Affinity chromatography (AC) Gel filtration (GF) Hydrophobic interaction (HIC), Reversed phase (RPC) Chromatofocusing

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

IEX chromatogram

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

AC chromatogram

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.

FG chromatogram

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

Technique Vs three phase

combinations of chromatographic steps

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.

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